Whole exome sequencing reveal 83 novel Mendelian disorders carrier P/LP variants in Chinese adult patients.

Carrier screening can identify people at risk of conceiving pregnancies affected with inherited genetic disorders or who have a genetic disorder with late or variable onset. Carrier screening based on whole exome sequencing (WES) data can offer more comprehensive assessment than on-target carrier screening tests. A total of 224 Chinese adult patients WES data was analyzed, except positive variants associated with the patients' major complaint, 378 pathogenic (P) and "likely pathogenic" (LP) variants from 175 adult patients were identified. Whole exome-wide frequency of carriers for Mendelian disorders in Chinese adult patients was about 78.13% in this study, which was lower than the previously reported carrier frequency in healthy population. Contrary to expectations, the number of P or LP variants did not increase with larger chromosome size or decrease with smaller chromosome size. Totally 83 novel P or LP variants were identified which could further expand the carrier variants spectrum of the Chinese population. GJB2: NM_004004.6:c.299_300delAT:p.His100fs*14 and C6:NM_000065.4:c.654T>A:p.Cys218* were found in two or more patients, which might be two underestimated carrier variants in Chinese population. We also found 9 late-onset or atypical symptoms autosomal/X-linked dominant Mendelian disorders causative genes, which were easily overlooked during pathogenicity analysis. These results can provide a strong basis for preventing and avoiding the prevalence rates of birth defects and reducing social and family burdens. By comparing with three different expanded carrier screening gene panels, we further confirmed carrier screening based on WES could offer more comprehensive assessment and WES was applicable for carrier screening.

Sleep Duration Positively Correlates with Global Cognition in the Non-Demented Older Adults with High School or above Education.

BACKGROUND: Sleep disturbance is common in the elderly. The effect of sleep duration on cognitive function in the non-demented older adults with high school or above education needs to be clarified. Here, we conducted a cross-sectional study to explore the correlation between sleep duration and multi-domain cognitive function in non-demented older adults. METHODS: A total of 226 adults aged 60 years and over who have an educational background over 9 years, received a battery of neuropsychological evaluations. The Mini-Mental State Examination (MMSE) was used to assess global cognitive function, the Auditory Verbal Learning Test (AVLT), Verbal Fluent Test (VFT), Trial Making Test-A/B (TMT-A/B), Symbol Digit Modalities Test (SDMT), and Rey-Osterriech Complex Figure Test (CFT) were used to assess the memory, language, attention and executive, and visuospatial functions respectively. Sleep characteristics were collected by questionnaire. RESULTS: Subjects with sleep disturbance performed worse in visuospatial ability as compared with those with normal sleep. A significant correlation between nocturnal/total sleep duration and MMSE scores and CFT scores was found in overall subjects using linear regression models after adjusting for age, gender, education and BMI. Consistently, the nocturnal/total sleep duration positively correlated with MMSE scores after controlling for age, gender, education, BMI, hypertension, diabetes, hyperlipidemia, coronary artery disease and household conditions. CONCLUSIONS: The results indicate that shorter sleep duration impairs the global cognition and visuospatial ability in the older adults with high school or above education, even in the very early non-demented stage.

Engineering Allosteric Ribozymes to Detect Thiamine Pyrophosphate in Whole Blood.

Thiamine deficiency contributes to several human diseases including Alzheimer's. As its biologically active form, thiamine pyrophosphate (TPP) has been considered as a potential biomarker for Alzheimer's disease (AD) based on several clinical reports that apparently lower blood TPP levels were found in patients with mild cognitive impairment to AD. However, highly sensitive and high-throughput detection of TPP in biological fluids remains an analytical challenge. Here, we report engineering RNA-based sensors to quantitatively measure TPP concentrations in whole blood samples with a detection limit down to a few nM. By fusing a TPP-specific aptamer with the hammerhead ribozyme for in vitro selection, we isolated an allosteric ribozyme with an EC50 value (68 nM) similar to the aptamer's KD value (50 nM) for TPP, which for the first time demonstrates the possibility to maintain the effector binding affinity of the aptamer in such engineered allosteric RNA constructs. Meanwhile, we developed a new blood sample preparation protocol to be compatible with RNA. By coupling the TPP-induced ribozyme cleavage event with isothermal amplification, we achieved fluorescence monitoring of whole blood TPP levels through the "mix-and-read" operation with high-throughput potential. We expect that the engineered TPP-sensing RNAs will facilitate clinical research on AD as well as other thiamine-related diseases.

Mild cognitive impairment in de novo Parkinson's disease: A neuromelanin MRI study in locus coeruleus.

BACKGROUND: We aimed to investigate neuromelanin-sensitive magnetic resonance imaging (NM-MRI) features in the locus coeruleus of de novo Parkinson's disease patients with different cognitive states and to determine whether these features are associated with cognitive impairment. METHODS: Three groups of subjects were recruited in this study, including patients with de novo PD with mild cognitive impairment (n = 23), patients with de novo PD without cognitive impairment (n = 48), and control subjects (n = 32). All subjects underwent clinical evaluations, as well as MRI scanning. The contrast-to-noise ratio of the locus coeruleus in the neuromelanin-sensitive MRI images and cortical thickness were measured. RESULTS: The contrast-to-noise ratio of the locus coeruleus in PD patients with mild cognitive impairment was significantly lower than that of controls (P = 0.016). The contrast-to-noise ratio of the locus coeruleus for PD patients without cognitive impairment was intermediate between that of controls and PD patients with mild cognitive impairment. Furthermore, multiple linear regression analysis showed that the contrast-to-noise ratio of the locus coeruleus was negatively associated with performance on the Trail Making Test B in all PD patients, controlling for age, sex, years of education, the Unified Parkinson's Disease Rating Scale motor scores from right upper limb, Geriatric Depression Rating Scales scores, Rapid Eye Movement Sleep Behavior Disorder Screening Questionnaire scores, and cortical thickness. CONCLUSIONS: Dysfunction of the locus coeruleus neurons may partly contribute to the decline in executive function in early de novo PD. In the future, the locus coeruleus-norepinephrine system might be targeted for early-intervention strategies in PD patients. © 2019 International Parkinson and Movement Disorder Society.

Thiamine deficiency contributes to synapse and neural circuit defects.

BACKGROUND: The previous studies have demonstrated the reduction of thiamine diphosphate is specific to Alzheimer's disease (AD) and causal factor of brain glucose hypometabolism, which is considered as a neurodegenerative index of AD and closely correlates with the degree of cognitive impairment. The reduction of thiamine diphosphate may contribute to the dysfunction of synapses and neural circuits, finally leading to cognitive decline. RESULTS: To demonstrate this hypothesis, we established abnormalities in the glucose metabolism utilizing thiamine deficiency in vitro and in vivo, and we found dramatically reduced dendrite spine density. We further detected lowered excitatory neurotransmission and impaired hippocampal long-term potentiation, which are induced by TPK RNAi in vitro. Importantly, via treatment with benfotiamine, Aß induced spines density decrease was considerably ameliorated. CONCLUSIONS: These results revealed that thiamine deficiency contributed to synaptic dysfunction which strongly related to AD pathogenesis. Our results provide new insights into pathogenesis of synaptic and neuronal dysfunction in AD.

MTERF2 contributes to MPP(+)-induced mitochondrial dysfunction and cell damage.

Parkinson's disease (PD) is a common neurodegenerative disorder whose pathogenesis is under intense investigation. Substantial evidence indicates that mitochondrial dysfunction plays a central role in the pathophysiology of PD. Several mitochondrial internal regulating factors act to maintain the mitochondrial function. However, how these internal regulating factors contribute to mitochondrial dysfunction in PD remains elusive. One of these factors, mitochondrial transcription termination factor 2 (MTERF2), has been implicated in the regulation of oxidative phosphorylation by modulating mitochondrial DNA transcription. Here, we discovered a new role of MTERF2 in regulating mitochondrial dysfunction and cell damage induced by MPP(+) in SH-SY5Y cells. We found that MPP(+) treatment elevated MTERF2 expression, induced mitochondrial dysfunction and cell damage, which was alleviated by MTERF2 knockdown. These findings demonstrate that MTERF2 contributes to MPP(+)-induced mitochondrial disruption and cell damage. This study indicates that MTERF2 is a potential therapeutic target for environmentally induced Parkinson's disease.

Rearrangement structure-independent strategy of CNV breakpoint analysis.

Rare copy number variations (CNVs) generated by human genomic rearrangements have been shown to play an important role in pathogenesis of human diseases and cancers. CNV breakpoint analysis can help define genomic location, genetic content and sequence structure of pathogenic CNVs. This process is vital to elucidate CNV mutational mechanism and etiology of CNV-associated disorders. However, it is technically challenging to map CNV breakpoints at base-pair level, especially in the genomic regions with sequence complexity. In this study, we developed a new method of capture and breakpoint approaching sequencing (CBAS) to efficiently obtain CNV breakpoint sequences. This strategy is independent of CNV structures and applicable to various CNV types. As was demonstrated in CNV-associated patients with neurological disorders, CBAS achieved fine mapping of breakpoint sequences for compound deletion, complex duplication, and translocation. Intriguingly, CBAS also revealed unexpected CNV complexity involving long-range DNA rearrangement. Our observations showed that CBAS is an efficient method for obtaining CNV breakpoint sequence and mapping insertional events as well. This method can facilitate the researches on CNV-associated human diseases and cancers. CBAS is also applicable to mapping the integration sites of retrovirus (such as HIV) and transgenes in model organisms.

Low expression of thiamine pyrophosphokinase-1 contributes to brain susceptibility to thiamine deficiency.

Thiamine deficiency is a well-known risk factor for the development of severe encephalopathy, such as Wernicke encephalopathy and Korsakoff syndrome, but the underlying mechanism is still mysterious. This study aims to investigate the expression levels of thiamine metabolism genes in different tissues and their impact on brain susceptibility to thiamine deficiency. The mRNA and protein levels of four genes known to be associated with thiamine metabolism: thiamine pyrophosphokinase-1 (Tpk), Solute carrier family 19 member 2 (Slc19a2), Slc19a3, and Slc25a19, in the brain, kidney, and liver of mice were examined. Thiamine diphosphate (TDP) levels were measured in these tissues. Mice were subjected to dietary thiamine deprivation plus pyrithiamine (PTD), a specific TPK inhibitor, or pyrithiamine alone to observe the reduction in TDP and associated pathological changes. TPK mRNA and protein expression levels were lowest in the brain compared to the kidney and liver. Correspondingly, TDP levels were also lowest in the brain. Mice treated with PTD or pyrithiamine alone showed an initial reduction in brain TDP levels, followed by reductions in the liver and kidney. PTD treatment caused significant neuron loss, neuroinflammation, and blood-brain barrier disruption, whereas dietary thiamine deprivation alone did not. TPK expression level is the best indicator of thiamine metabolism status. Low TPK expression in the brain appears likely to contribute to brain susceptibility to thiamine deficiency, underscoring a critical role of TPK in maintaining cerebral thiamine metabolism and preventing thiamine deficiency-related brain lesions.

Identification of biomarkers differentiating Alzheimer's disease from other neurodegenerative diseases by integrated bioinformatic analysis and machine-learning strategies.

Background: Alzheimer's disease (AD) is the most common neurodegenerative disease, imposing huge mental and economic burdens on patients and society. The specific molecular pathway(s) and biomarker(s) that distinguish AD from other neurodegenerative diseases and reflect the disease progression are still not well studied. Methods: Four frontal cortical datasets of AD were integrated to conduct differentially expressed genes (DEGs) and functional gene enrichment analyses. The transcriptional changes after the integrated frontal cortical datasets subtracting the cerebellar dataset of AD were further compared with frontal cortical datasets of frontotemporal dementia and Huntingdon's disease to identify AD-frontal-associated gene expression. Integrated bioinformatic analysis and machine-learning strategies were applied for screening and determining diagnostic biomarkers, which were further validated in another two frontal cortical datasets of AD by receiver operating characteristic (ROC) curves. Results: Six hundred and twenty-six DEGs were identified as AD frontal associated, including 580 downregulated genes and 46 upregulated genes. The functional enrichment analysis revealed that immune response and oxidative stress were enriched in AD patients. Decorin (DCN) and regulator of G protein signaling 1 (RGS1) were screened as diagnostic biomarkers in distinguishing AD from frontotemporal dementia and Huntingdon's disease of AD. The diagnostic effects of DCN and RGS1 for AD were further validated in another two datasets of AD: the areas under the curve (AUCs) reached 0.8148 and 0.8262 in GSE33000, and 0.8595 and 0.8675 in GSE44770. There was a better value for AD diagnosis when combining performances of DCN and RGS1 with the AUCs of 0.863 and 0.869. Further, DCN mRNA level was correlated to CDR (Clinical Dementia Rating scale) score (r = 0.5066, p = 0.0058) and Braak staging (r = 0.3348, p = 0.0549). Conclusion: DCN and RGS1 associated with the immune response may be useful biomarkers for diagnosing AD and distinguishing the disease from frontotemporal dementia and Huntingdon's disease. DCN mRNA level reflects the development of the disease.

Physiological Roles of ß-amyloid in Regulating Synaptic Function: Implications for AD Pathophysiology.

The physiological functions of endogenous amyloid-ß (Aß), which plays important role in the pathology of Alzheimer's disease (AD), have not been paid enough attention. Here, we review the multiple physiological effects of Aß, particularly in regulating synaptic transmission, and the possible mechanisms, in order to decipher the real characters of Aß under both physiological and pathological conditions. Some worthy studies have shown that the deprivation of endogenous Aß gives rise to synaptic dysfunction and cognitive deficiency, while the moderate elevation of this peptide enhances long term potentiation and leads to neuronal hyperexcitability. In this review, we provide a new view for understanding the role of Aß in AD pathophysiology from the perspective of physiological meaning.

A comparison of the five-minute cognitive test with the mini-mental state examination in the elderly for cognitive impairment screening.

The five-minute cognitive test (FCT) is a novel cognitive screening method with the quick and reliable merit for detecting cognitive impairment at an early stage. The diagnostic power of FCT in differentiating subjects with cognitive impairment from people with cognition in a normal range was demonstrated effective as that of the Mini-Mental Status Evaluation (MMSE) in a previous cohort study. Here, we analyzed the effect of sociodemographic and health-related factors on FCT performance and further investigated the consistency of FCT. Then, we compared the correlation of subitem scores of FCT or MMSE with a comprehensive battery of neuropsychological tests that focus on specific domains of cognition. Finally, the association of the total FCT scores with the volumes of brain subregions was investigated. There were 360 subjects aged 60 years or above enrolled in this study, including 226 adults with cognitive abilities in normal range, 107 subjects with mild cognitive impairment (MCI) and 27 mild Alzheimer's disease (AD). The results showed that the total FCT scores was negatively associated with increasing age (ß = -0.146, p < 0.001), and positively associated with education attainment (ß = 0.318, p < 0.001), dwelling condition with family (ß = 0.153, p < 0.001) and the Body Mass Index (ß = 1.519, p < 0.01). The internal consistency of the FCT (Cronbach's α) was 0.644. The sub-scores of FCT showed a significant correlation with other specific neuropsychological tests. Impressively, the total FCT scores showed a significantly positive association with the volumes of hippocampus related subregions (r = 0.523, p < 0.001) and amygdala (r = 0.479, p < 0.001), but not with cerebellum (r = 0.158, p > 0.05) or subcortical subregions (r = 0.070, p > 0.05). Combining with previous data, FCT is a reliable and valid cognitive screening test for detecting cognitive impairment in a community setting.

Identification of diagnostic biomarkers in Alzheimer's disease by integrated bioinformatic analysis and machine learning strategies.

Background: Alzheimer's disease (AD) is the most prevalent form of dementia, and is becoming one of the most burdening and lethal diseases. More useful biomarkers for diagnosing AD and reflecting the disease progression are in need and of significance. Methods: The integrated bioinformatic analysis combined with machine-learning strategies was applied for exploring crucial functional pathways and identifying diagnostic biomarkers of AD. Four datasets (GSE5281, GSE131617, GSE48350, and GSE84422) with samples of AD frontal cortex are integrated as experimental datasets, and another two datasets (GSE33000 and GSE44772) with samples of AD frontal cortex were used to perform validation analyses. Functional Correlation enrichment analyses were conducted based on Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Reactome database to reveal AD-associated biological functions and key pathways. Four models were employed to screen the potential diagnostic biomarkers, including one bioinformatic analysis of Weighted gene co-expression network analysis (WGCNA)and three machine-learning algorithms: Least absolute shrinkage and selection operator (LASSO), support vector machine-recursive feature elimination (SVM-RFE) and random forest (RF) analysis. The correlation analysis was performed to explore the correlation between the identified biomarkers with CDR scores and Braak staging. Results: The pathways of the immune response and oxidative stress were identified as playing a crucial role during AD. Thioredoxin interacting protein (TXNIP), early growth response 1 (EGR1), and insulin-like growth factor binding protein 5 (IGFBP5) were screened as diagnostic markers of AD. The diagnostic efficacy of TXNIP, EGR1, and IGFBP5 was validated with corresponding AUCs of 0.857, 0.888, and 0.856 in dataset GSE33000, 0.867, 0.909, and 0.841 in dataset GSE44770. And the AUCs of the combination of these three biomarkers as a diagnostic tool for AD were 0.954 and 0.938 in the two verification datasets. Conclusion: The pathways of immune response and oxidative stress can play a crucial role in the pathogenesis of AD. TXNIP, EGR1, and IGFBP5 are useful biomarkers for diagnosing AD and their mRNA level may reflect the development of the disease by correlation with the CDR scores and Breaking staging.

The Dynamic SUMOylation Changes and Their Potential Role in the Senescence of APOE4 Mice.

The ε4 allele of apolipoprotein E (APOE4) and aging are the major risk factors for Alzheimer's disease (AD). SUMOylation is intimately linked to the development of AD and the aging process. However, the SUMOylation status in APOE4 mice has not been uncovered. In this study, we investigated SENP1 and SUMOylation changes in the brains of aged APOE3 and APOE4 mice, aiming to understand their potential impact on mitochondrial metabolism and their contribution to cellular senescence in APOE4 mice. Concurrently, SUMO1-conjugated protein levels decreased, while SUMO2/3-conjugated protein levels increased relatively with the aging of APOE4 mice. This suggests that the equilibrium between the SUMOylation and deSUMOylation processes may be associated with senescence and longevity. Our findings highlight the significant roles of SENP1 and SUMOylation changes in APOE4-driven pathology and the aging process.

Intronic position +9 and -9 are potentially splicing sites boundary from intronic variants analysis of whole exome sequencing data.

Whole exome sequencing (WES) can also detect some intronic variants, which may affect splicing and gene expression, but how to use these intronic variants, and the characteristics about them has not been reported. This study aims to reveal the characteristics of intronic variant in WES data, to further improve the clinical diagnostic value of WES. A total of 269 WES data was analyzed, 688,778 raw variants were called, among these 367,469 intronic variants were in intronic regions flanking exons which was upstream/downstream region of the exon (default is 200 bps). Contrary to expectation, the number of intronic variants with quality control (QC) passed was the lowest at the +2 and -2 positions but not at the +1 and -1 positions. The plausible explanation was that the former had the worst effect on trans-splicing, whereas the latter did not completely abolish splicing. And surprisingly, the number of intronic variants that passed QC was the highest at the +9 and -9 positions, indicating a potential splicing site boundary. The proportion of variants which could not pass QC filtering (false variants) in the intronic regions flanking exons generally accord with "S"-shaped curve. At +5 and -5 positions, the number of variants predicted damaging by software was most. This was also the position at which many pathogenic variants had been reported in recent years. Our study revealed the characteristics of intronic variant in WES data for the first time, we found the +9 and -9 positions might be a potentially splicing sites boundary and +5 and -5 positions were potentially important sites affecting splicing or gene expression, the +2 and -2 positions seem more important splicing site than +1 and -1 positions, and we found variants in intronic regions flanking exons over ± 50 bps may be unreliable. This result can help researchers find more useful variants and demonstrate that WES data is valuable for intronic variants analysis.

Decreased serum ceruloplasmin levels characteristically aggravate nigral iron deposition in Parkinson's disease.

In vivo and post-mortem studies have demonstrated that increased nigral iron content in patients with Parkinson's disease is a prominent pathophysiological feature. However, the mechanism and risk factors associated with nigral iron deposition in patients with Parkinson's disease have not been identified and represent a key challenge in understanding its pathogenesis and for its diagnosis. In this study, we assessed iron levels in patients with Parkinson's disease and in age- and gender-matched control subjects by measuring phase values using magnetic resonance based susceptibility-weighted phase imaging in a 3T magnetic resonance system. Phase values were measured from brain regions including bilateral substantia nigra, globus pallidus, putamen, caudate, thalamus, red nucleus and frontal white matter of 45 patients with Parkinson's disease with decreased or normal serum ceruloplasmin levels, together with age- and gender-matched control subjects. Correlative analyses between phase values, serum ceruloplasmin levels and disease severity showed that the nigral bilateral average phase values in patients with Parkinson's disease were significantly lower than in control subjects and correlated with disease severity according to the Hoehn and Yahr Scale. The Unified Parkinson's Disease Rating Scale motor scores from the clinically most affected side were significantly correlated with the phase values of the contralateral substantia nigra. Furthermore, nigral bilateral average phase values correlated highly with the level of serum ceruloplasmin. Specifically, in the subset of patients with Parkinson's disease exhibiting reduced levels of serum ceruloplasmin, we found lowered nigral bilateral average phase values, suggesting increased nigral iron content, while those patients with normal levels of serum ceruloplasmin exhibited no changes as compared with control subjects. These findings suggest that decreased levels of serum ceruloplasmin may specifically exacerbate nigral iron deposition in patients with Parkinson's disease. Combining susceptibility-weighted phase imaging with serum ceruloplasmin determination is likely to be useful for the diagnosis and assessment of a subset of patients with Parkinson's disease.

Association Between Blood Biochemical Factors Contributing to Cognitive Decline and B Vitamins in Patients With Alzheimer's Disease.

Background: Malnutrition, metabolism stress, inflammation, peripheral organs dysfunction, and B vitamins deficiency significantly contribute to the progression and mortality of Alzheimer's disease (AD). However, it is unclear which blood biochemical indicators are most closely related to cognitive decline and B vitamins deficiency (thiamine, folate, vitamin B12) in patients with AD. Methods: This was a cross-sectional study of 206 AD patients recruited from six hospitals in China. Thiamine diphosphate (TDP), the bioactive form of thiamine, was measured by high-performance liquid chromatography fluoroscopy (HPLC) at a single center. Levels of biochemical indicators (except TDP) were measured by regular and standard laboratory tests in each hospital. Pearson's rank correlation analysis was used to assess relationships between B vitamins and biochemical indicators. T-test was used to compare the difference between ApoE ε4 and non-ApoE ε4 groups. Differences were considered statistically significant as P < 0.05. Results: Among the biochemical results, in AD population, malnutrition indicators (erythrocyte, hemoglobin, serum albumin, and total protein) were most significantly associated with cognitive function, as was free triiodothyronine (FT3) levels which had been observed in previous study. Malnutrition and FT3 levels depend on age but not apolipoprotein E (ApoE) genotype. Meanwhile, Among the B vitamins, TDP was the most significantly associated with malnutrition indicators and FT3. Conclusion: Our results indicated that TDP reduction could be a modifiable risk factor for malnutrition and FT3 that contributed to cognitive decline in AD patients. Correcting thiamine metabolism could serve as an optional therapy target for AD treatment.

Powerful beneficial effects of benfotiamine on cognitive impairment and beta-amyloid deposition in amyloid precursor protein/presenilin-1 transgenic mice.

Reduction of glucose metabolism in brain is one of the main features of Alzheimer's disease. Thiamine (vitamin B1)-dependent processes are critical in glucose metabolism and have been found to be impaired in brains from patients with Alzheimer's disease. However, thiamine treatment exerts little beneficial effect in these patients. Here, we tested the effect of benfotiamine, a thiamine derivative with better bioavailability than thiamine, on cognitive impairment and pathology alterations in a mouse model of Alzheimer's disease, the amyloid precursor protein/presenilin-1 transgenic mouse. We show that after a chronic 8 week treatment, benfotiamine dose-dependently enhanced the spatial memory of amyloid precursor protein/presenilin-1 mice in the Morris water maze test. Furthermore, benfotiamine effectively reduced both amyloid plaque numbers and phosphorylated tau levels in cortical areas of the transgenic mice brains. Unexpectedly, these effects were not mimicked by another lipophilic thiamine derivative, fursultiamine, although both benfotiamine and fursultiamine were effective in increasing the levels of free thiamine in the brain. Most notably, benfotiamine, but not fursultiamine, significantly elevated the phosphorylation level of glycogen synthase kinase-3alpha and -3beta, and reduced their enzymatic activities in the amyloid precursor protein/presenilin-1 transgenic brain. Therefore, in the animal Alzheimer's disease model, benfotiamine appears to improve the cognitive function and reduce amyloid deposition via thiamine-independent mechanisms, which are likely to include the suppression of glycogen synthase kinase-3 activities. These results suggest that, unlike many other thiamine-related drugs, benfotiamine may be beneficial for clinical Alzheimer's disease treatment.

The impact of thiamine deficiency and benfotiamine treatment on Nod-like receptor protein-3 inflammasome in microglia.

Thiamine-dependent processes are critical in cerebral glucose metabolism, it is abnormity induces oxidative stress, inflammation and neurodegeneration. Nod-like receptor protein-3 (NLRP3) inflammasome-mediated inflammation is closely related to neurologic diseases and can be activated by oxidative stress. However, the impact of thiamine deficiency on NLRP3 inflammasome activation remains unknown. In this study, we found that NLRP3 inflammasomes were significantly activated in the microglia of thiamine deficiency mice model. In contrast, benfotiamine dampened inflammation NLRP3 mediated in BV2 cells stimulated with LPS and ATP through reducing mitochondrial reactive oxygen species levels and mitigating autophagy flux defect. These data identify an important role of thiamine metabolism in NLRP3 inflammasome activation, and correcting thiamine metabolism through benfotiamine provides a new therapeutic strategy for NLRP3 inflammasome related neurological, metabolic, and inflammatory diseases.

Dynamic Change of Intracellular Metabolism of Microglia Evaluated by Transcriptomics in an Alzheimer's Mouse Model.

BACKGROUND: Microglia play diverse roles in Alzheimer's disease (AD). Intracellular metabolism has been indicated an important factor in modulating the function of microglia. However, it is not clear whether the intracellular metabolism of microglia changes dynamically in different stages of AD. OBJECTIVE: To determine whether microglia intracellular metabolism changes dynamically in different stages of AD. METHODS: Microglia were extracted from APPSwe/PS1dE9 (APP/PS1) mice and wild-type littermates at 2, 4, and 8 months old by fluorescence-activated cell sorting and used for RNA-sequencing analysis and quantitative PCR. Morphologies of amyloid plaques and microglia were detected by immunofluorescence staining. RESULTS: Compared with control littermates, the microglia of APP/PS1 mice exhibited significant transcriptional changes at 2-month-old before microglia morphological alterations and the plaque formation. The changes continued drastically following age with defined morphological shift of microglia and amyloid plaque enhancement in brains. Further analysis of those genotype and age dependent transcriptomic changes revealed that differentially expressed genes were enriched in pathways related to energy metabolism. Compared with wild-type mice, there were changes of some vital genes related to glucose metabolism and lipid metabolism pathways in APP/PS1 mice at different ages. Glucose metabolism may play a major role in early activation of microglia, and lipid metabolism may be more important in later activation period. CONCLUSION: Our results showed that microglia actively participate in the pathological progress of AD. The intracellular metabolism of microglia changed significantly in different stages of AD, even preceding amyloid-ß deposition.

Safety, Tolerability and Pharmacokinetics of Single and Multiple Ascending Doses of Benfotiamine in Healthy Subjects.

PURPOSE: Safety, tolerability and pharmacokinetics of single and multiple ascending doses (SADs/MADs) of benfotiamine were assessed after oral administration in two randomized, double-blind, placebo-controlled, phase I trials. METHODS: Healthy subjects were sequentially enrolled into one of five SAD (150-1200 mg) or three MAD (150, 300 or 600 mg) cohorts. In SAD study, each cohort of 12 subjects (n = 10, active; n = 2, placebo) were administrated once-daily doses. In MAD study, each cohort of 16 subjects (n = 12, active; n = 4, placebo) were administrated once-daily on day 1 and twice-daily on day 4-9, followed by a single morning dose on day 10. RESULTS: In the SAD study, the median time to reach maximum concentration (Tmax) arrived 1.0 to 2.0 h for thiamine (TM), 3.5 to 8.0 h for thiamine monophosphate (TMP), and 8.0 to 24.0 h for thiamine diphosphate (TDP) after administration of benfotiamine. The area under concentration-time curve from 0 to last measurable concentration (AUC0-t) or maximum observed concentration (Cmax) of TM, TMP, and TDP was less or more dose proportional over the single dose studied except Cmax of TM. Food consumption did not increase the level of TM and TDP at baseline. TM exhibited a relatively long elimination half-life (t1/2) in all doses studied, resulting in accumulation ratio (Rac) of 1.96 to 2.11 and accumulation ratio based on Cmax (Rac, Cmax) of 1.60 to 1.88 following 7 days of multiple dosing. Comparable accumulation results were also obtained for TDP after multiple dosing. The incidence and severity of adverse events (AEs) were similar between benfotiamine and placebo. The commonly reported drug-related AEs were increased ALT and urinary WBC. CONCLUSION: Both SAD and MAD studies of benfotiamine in healthy subjects were safe and well tolerated. TM and TDP exhibited moderate accumulation on repeated administration of benfotiamine.