CHRISTMAS: Chiral Pair Isobaric Labeling Strategy for Multiplexed Absolute Quantitation of Enantiomeric Amino Acids.

Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer's disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.

Increased expression of SLC25A1/CIC causes an autistic-like phenotype with altered neuron morphology.

N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.

A commitment to marginalized children in the European Union: The hope and challenges of the EU Child Guarantee.

While children in general are usually seen as a societal priority, many children are disadvantaged by marginalization, with adverse effects on health and development. Following feasibility studies, the European Commission has now adopted a formal Child Guarantee of service access. This paper links the Feasibility Studies to other reports on the need to address marginalized and institutionalized children. The problems in identifying and quantifying such children are outlined, as are the challenges of planning for these groups of children and the difficulty of finding universal definitions and data. This European Union initiative is timely, given that around a quarter of European children are marginalized, while the effects of Covid-19 will add to this marginalization.

European Union Child Guarantee-challenges raised by the welcome promise of free healthcare for marginalized children.

BACKGROUND: Children are dependent on the way in which society provides healthcare, with primary and preventive care being initial components. They also have a generally acclaimed right to health, and to lack of impediment to access to healthcare. In a major initiative, the European Parliament has proposed a Child Guarantee to include free access to healthcare for marginalized children, and a Feasibility Study has been completed with positive results. However, there has been little analysis of national policies toward free access to healthcare for children, including longer-term treatment, mental health or adolescent health services, or of charges and indirect financial barriers to access. METHODS: Data on policies for children's access to healthcare from two recent European Community wide studies were re-analyzed and matched. Primary care, immunization, surveillance screening, minor illness, a more significant medium-term condition, and reproductive health were included. Additionally, data from a European survey of children reported as having unmet medical needs were revisited. Composite summaries relating to all 28 EU countries as of 2019 were produced. RESULTS: Only three EU-28 countries provided totally free services, though 26 countries provide free primary and preventive services. There is evidence of some children having unmet medical needs in 21 countries, with Expense being the main quoted factor. CONCLUSION: There is widespread variation across Europe in free access for children to healthcare, little comparative study of policies and their effects on enabling or hindering access, and minimal data collection. This compromises achievement of the Guarantee, and initiatives are needed.

After the European Global Vaccination Summit-The need for practical policies to boost vaccination delivery to children in Europe.

BACKGROUND: Low childhood vaccination rates in Europe continue to cause concern and have triggered much policy study. However, regarding children, making immunisation a stand-alone issue cuts across integrated child health services. Most initiatives, including the 10 Action Points from the 2019 European-hosted Global Vaccination Summit, are all laudable but high level enablement policies. Delivery processes have been ignored, and key stakeholders not involved in discussions. METHODS: Reviews of policies, literature and planned actions, leading to identification of further delivery policies needed to facilitate and stimulate practical accomplishments. RESULTS: 10 aspects are identified where European coordinated action to develop policies, share evidence and increase standards, would be beneficial. These also fit in with established European Commission strengths, and the incoming Commission's policies and priorities. CONCLUSION: The European Commission, Member States and child health stakeholders should pursue a holistic approach, taking immunisation as a component of integrated child health. Service delivery should be compatible with modern societal challenges and related parenting patterns, and public health processes modernised and reinvigorated. Nursing and societal stakeholders should be brought in, and fit-for-purpose digital support facilitated. This is even more urgent following the diversion of the Covid-19 pandemic.

The endoplasmic reticulum acetyltransferases ATase1/NAT8B and ATase2/NAT8 are differentially regulated to adjust engagement of the secretory pathway.

Nε-lysine acetylation of nascent glycoproteins within the endoplasmic reticulum (ER) lumen regulates the efficiency of the secretory pathway. The ER acetylation machinery consists of the membrane transporter, acetyl-CoA transporter 1 (AT-1/SLC33A1), and two acetyltransferases, ATase1/NAT8B and ATase2/NAT8. Dysfunctional ER acetylation is associated with severe neurological diseases with duplication of AT-1/SLC33A1 being associated with autism spectrum disorder, intellectual disability, and dysmorphism. Neuron-specific AT-1 over-expression in the mouse alters neuron morphology and function, causing an autism-like phenotype, indicating that ER acetylation plays a key role in neurophysiology. As such, characterizing the molecular mechanisms that regulate the acetylation machinery could reveal critical information about its biology. By using structure-biochemistry approaches, we discovered that ATase1 and ATase2 share enzymatic properties but differ in that ATase1 is post-translationally regulated via acetylation. Furthermore, gene expression studies revealed that the promoters of AT-1, ATase1, and ATase2 contain functional binding sites for the neuron-related transcription factors cAMP response element-binding protein and the immediate-early genes c-FOS and c-JUN, and that ATase1 and ATase2 exhibit additional modes of transcriptional regulation relevant to aging and Alzheimer's disease. In vivo rodent gene expression experiments revealed that Atase2 is specifically induced following activity-dependent events. Finally, over-expression of either ATase1 or ATase2 was sufficient to increase the engagement of the secretory pathway in PC12 cells. Our results indicate important regulatory roles for ATase1 and ATase2 in neuron function with induction of ATase2 expression potentially serving as a critical event that adjusts the efficiency of the secretory pathway for activity-dependent neuronal functions.

Child health research and planning in Europe disadvantaged by major gaps and disparities in published statistics.

BACKGROUND: Population data, such as mortality and morbidity statistics, are essential for many reasons, including giving context for research, supporting action on health determinants, formulation of evidence-based policy for health care and outcome evaluation. However, when considering children, it is difficult to find such data, despite children comprising one-fifth of the European population and being in a key formative life stage and dependent on societal support. Moreover, it would be expected that there should be confidence in the key child health data available, with little to no discrepancy between recognized health statistic databases. METHODS: This study explored the main health databases in or including Europe to collate child mortality data, for both all-cause and specific-cause mortality. Tables were constructed for comparison of values and rankings. RESULTS: The results show that there are major differences in reported mortality data between two prominent health statistic databases, difference in coding systems, and unannounced changes within one of the databases. CONCLUSIONS: The lack of health data for children seems compounded by challenges to the trust and credibility, which are vital if these data are to have utility. Children and society are the losers, and resolution is needed as a priority.

European Union initiatives in child immunization-the need for child centricity, e-health and holistic delivery.

BACKGROUND: Low childhood immunization rates in Europe are causing concern and have triggered several EU initiatives. However, these are counter-factual as they make immunization a stand-alone issue and cut across best practice in integrated child health services. They also focus unduly on 'anti-vax' pressures, generalize 'vaccine hesitancy' and overlook practical difficulties and uncertainties encountered by parents in real world situations about presenting children for immunization. Meanwhile European expertize in child health electronic record systems and relevant standards are ignored despite their being a potentially sound foundation ripe for enhancement. METHODS: Situation and literature reviews, and cohesion of two European research projects, led to shared investigation. As a result, two cross-sectoral expert workshops were held to consider digital health standards for harmonizing integrated preventive child health including immunization, and the work of other stakeholders such as the World Health Organisation and the European Centre for Disease Control. RESULTS: Progress in child health information models and digital health standards was assessed, areas needing further standards development identified and desirable steps towards innovation in service delivery and record keeping agreed. CONCLUSION: The European Commission, member states and child health stakeholders should take an integrated approach to child health with immunization as a component. Service delivery should be sensitive to parental concerns and challenges, and the way child- and family-centric data are recorded and used should be enhanced. Services should be enabled by the International Patient Summary and related electronic health record standards and linkages, and evaluated to assess most effective systems and practice.

Potentially over 3 million children in EU Europe believed not to be receiving needed medical and dental treatment-and parents' reasons why.

BACKGROUND: Children have the right to health and countries a duty under the United Nations Convention on the Rights of the Child to facilitate this. The European Union has emphasized the importance of investing in children, but at times this seems more wish than pragmatism. Furthermore, European statistical systems do not provide any relevant data, and the degree of unmet need has hitherto been unknown. However, new ad hoc household survey data have now been published by Eurostat showing the percentage of children with a purported unmet medical or dental need and the expressed reasons for this. METHOD: This paper critically reviews these data on children with a reported unmet medical or dental need to create an indication of the number of European children with unmet medical and dental needs, and the contributory factors. RESULTS: This paper calculates that some 1 million European children can be estimated to have an unmet medical need and 2 million children an unmet dental need, though the survey approach has some weaknesses. A probable overestimate of children affected in sample households offsets the likely failure to capture data about children in institutions, homeless, or in fractured families, or about multiple needs. The reported reasons for not obtaining treatments are a valuable first step in highlighting an important issue for Europe's children-measurement of service accessibility. CONCLUSION: Potentially over 3 million European Union children are failing to have their health needs and their rights met. If the incoming European Commission is serious about its predecessor's promise to invest in children and to take seriously their rights, action is needed to improve quantification of unmet need and to reduce suffering and potential lasting damage.

Spatially and temporally probing distinctive glycerophospholipid alterations in Alzheimer's disease mouse brain via high-resolution ion mobility-enabled sn-position resolved lipidomics.

Dysregulated glycerophospholipid (GP) metabolism in the brain is associated with the progression of neurodegenerative diseases including Alzheimer's disease (AD). Routine liquid chromatography-mass spectrometry (LC-MS)-based large-scale lipidomic methods often fail to elucidate subtle yet important structural features such as sn-position, hindering the precise interrogation of GP molecules. Leveraging high-resolution demultiplexing (HRdm) ion mobility spectrometry (IMS), we develop a four-dimensional (4D) lipidomic strategy to resolve GP sn-position isomers. We further construct a comprehensive experimental 4D GP database of 498 GPs identified from the mouse brain and an in-depth extended 4D library of 2500 GPs predicted by machine learning, enabling automated profiling of GPs with detailed acyl chain sn-position assignment. Analyzing three mouse brain regions (hippocampus, cerebellum, and cortex), we successfully identify a total of 592 GPs including 130 pairs of sn-position isomers. Further temporal GPs analysis in the three functional brain regions illustrates their metabolic alterations in AD progression.

miR-92a-3p and miR-320a are Upregulated in Plasma Neuron-Derived Extracellular Vesicles of Patients with Frontotemporal Dementia.

Despite the efforts to identify fluid biomarkers to improve diagnosis of Frontotemporal dementia (FTD), only a few candidates have been described in recent years. In a previous study, we identified three circulating miRNAs (miR-92a-3p, miR-320a and miR-320b) differentially expressed in FTD patients with respect to healthy controls and/or Alzheimer's disease (AD) patients. Now, we investigated whether those changes could be due to miRNAs contained in neuron-derived extracellular vesicles (NDEVs). We also evaluated miRNAs content in total plasma EVs and in CSF samples. The analysis of plasma NDEVs carried out on 40 subjects including controls (n = 13), FTD (n = 13) and AD (n = 14) patients, showed that both miR-92a-3p and miR-320a levels were triplicated in the FTD group if compared with CT and AD patients. Increased levels of the same miRNAs were found also in CSF derived from FTD group compared to CTs. No differences were observed in expression levels of miR-320b among the three groups. Worthy of note, all miRNAs analysed were increased in an FTD cell model, MAPT IVS10 + 16 neurons. Our results suggest that miR-92a and miR-320a in NDEVs could be proposed as FTD biomarkers.

Acarbose ameliorates Western diet-induced metabolic and cognitive impairments in the 3xTg mouse model of Alzheimer's disease.

Age is the greatest risk factor for Alzheimer's disease (AD) as well as for other disorders that increase the risk of AD such as diabetes and obesity. There is growing interest in determining if interventions that promote metabolic health can prevent or delay AD. Acarbose is an anti-diabetic drug that not only improves glucose homeostasis, but also extends the lifespan of wild-type mice. Here, we test the hypothesis that acarbose will not only preserve metabolic health, but also slow or prevent AD pathology and cognitive deficits in 3xTg mice, a model of AD, fed either a Control diet or a high-fat, high-sucrose Western diet (WD). We find that acarbose decreases the body weight and adiposity of WD-fed 3xTg mice, increasing energy expenditure while also stimulating food consumption, and improves glycemic control. Both male and female WD-fed 3xTg mice have worsened cognitive deficits than Control-fed mice, and these deficits are ameliorated by acarbose treatment. Molecular and histological analysis of tau and amyloid pathology identified sex-specific effects of acarbose which are uncoupled from the dramatic improvements in cognition in females, suggesting that the benefits of acarbose on AD may be largely driven by improved metabolic health. In conclusion, our results suggest that acarbose may be a promising intervention to prevent, delay, or even treat AD, especially in individuals consuming a WD.

Acarbose ameliorates Western diet-induced metabolic and cognitive impairments in the 3xTg mouse model of Alzheimer's disease.

Age is the greatest risk factor for Alzheimer's disease (AD) as well as for other disorders that increase the risk of AD such as diabetes and obesity. There is growing interest in determining if interventions that promote metabolic health can prevent or delay AD. Acarbose is an anti-diabetic drug that not only improves glucose homeostasis, but also extends the lifespan of wild-type mice. Here, we test the hypothesis that acarbose will not only preserve metabolic health, but also slow or prevent AD pathology and cognitive deficits in 3xTg mice, a model of AD, fed either a Control diet or a high-fat, high-sucrose Western diet (WD). We find that acarbose decreases the body weight and adiposity of WD-fed 3xTg mice, increasing energy expenditure while also stimulating food consumption, and improves glycemic control. Both male and female WD-fed 3xTg mice have worsened cognitive deficits than Control-fed mice, and these deficits are ameliorated by acarbose treatment. Molecular and histological analysis of tau and amyloid pathology identified sex-specific effects of acarbose which are uncoupled from the dramatic improvements in cognition, suggesting that the benefits of acarbose on AD are largely driven by improved metabolic health. In conclusion, our results suggest that acarbose may be a promising intervention to prevent, delay, or even treat AD, especially in individuals consuming a Western diet.

Fasting is required for many of the benefits of calorie restriction in the 3xTg mouse model of Alzheimer's disease.

Caloric restriction (CR) is a widely recognized geroprotective intervention that slows or prevents Alzheimer's disease (AD) in animal models. CR is typically implemented via feeding mice a single meal per day; as CR mice rapidly consume their food, they are subject to a prolonged fast between meals. While CR has been shown to improve metabolic and cognitive functions and suppress pathological markers in AD mouse models, the specific contributions of fasting versus calorie reduction remains unclear. Here, we investigated the contribution of fasting and energy restriction to the beneficial effects of CR on AD progression. To test this, we placed 6-month-old 3xTg mice on one of several diet regimens, allowing us to dissect the effects of calories and fasting on metabolism, AD pathology, and cognition. We find that energy restriction alone, without fasting, was sufficient to improve glucose tolerance and reduce adiposity in both sexes, and to reduce Aß plaques and improve aspects of cognitive performance in females. However, we find that a prolonged fast between meals is necessary for many of the benefits of CR, including improved insulin sensitivity, reduced phosphorylation of tau, decreased neuroinflammation, inhibition of mTORC1 signaling, and activation of autophagy, as well as for the full cognitive benefits of CR. Finally, we find that fasting is essential for the benefits of CR on survival in male 3xTg mice. Overall, our results demonstrate that fasting is required for the full benefits of a CR diet on the development and progression of AD in 3xTg mice, and suggest that both when and how much we eat influences the development and progress of AD.

Protein restriction slows the development and progression of pathology in a mouse model of Alzheimer's disease.

Dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and dietary protein restriction extends the lifespan and healthspan of mice. In this study, we examined the effect of protein restriction (PR) on metabolic health and the development and progression of Alzheimer's disease (AD) in the 3xTg mouse model of AD. Here, we show that PR promotes leanness and glycemic control in 3xTg mice, specifically rescuing the glucose intolerance of 3xTg females. PR induces sex-specific alterations in circulating and brain metabolites, downregulating sphingolipid subclasses in 3xTg females. PR also reduces AD pathology and mTORC1 activity, increases autophagy, and improves the cognition of 3xTg mice. Finally, PR improves the survival of 3xTg mice. Our results suggest that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD.

Intracellular Citrate/acetyl-CoA flux and endoplasmic reticulum acetylation: Connectivity is the answer.

BACKGROUND: Key cellular metabolites reflecting the immediate activity of metabolic enzymes as well as the functional metabolic state of intracellular organelles can act as powerful signal regulators to ensure the activation of homeostatic responses. The citrate/acetyl-CoA pathway, initially recognized for its role in intermediate metabolism, has emerged as a fundamental branch of this nutrient-sensing homeostatic response. Emerging studies indicate that fluctuations in acetyl-CoA availability within different cellular organelles and compartments provides substrate-level regulation of many biological functions. A fundamental aspect of these regulatory functions involves Nε-lysine acetylation. SCOPE OF REVIEW: Here, we will examine the emerging regulatory functions of the citrate/acetyl-CoA pathway and the specific role of the endoplasmic reticulum (ER) acetylation machinery in the maintenance of intracellular crosstalk and homeostasis. These functions will be analyzed in the context of associated human diseases and specific mouse models of dysfunctional ER acetylation and citrate/acetyl-CoA flux. A primary objective of this review is to highlight the complex yet integrated response of compartment- and organelle-specific Nε-lysine acetylation to the intracellular availability and flux of acetyl-CoA, linking this important post-translational modification to cellular metabolism. MAJOR CONCLUSIONS: The ER acetylation machinery regulates the proteostatic functions of the organelle as well as the metabolic crosstalk between different intracellular organelles and compartments. This crosstalk enables the cell to impart adaptive responses within the ER and the secretory pathway. However, it also enables the ER to impart adaptive responses within different cellular organelles and compartments. Defects in the homeostatic balance of acetyl-CoA flux and ER acetylation reflect different but converging disease states in humans as well as converging phenotypes in relevant mouse models. In conclusion, citrate and acetyl-CoA should not only be seen as metabolic substrates of intermediate metabolism but also as signaling molecules that direct functional adaptation of the cell to both intracellular and extracellular messages. Future discoveries in CoA biology and acetylation are likely to yield novel therapeutic approaches.

Protein restriction slows the development and progression of Alzheimer's disease in mice.

Over the last decade, it has become evident that dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and we and others have shown that dietary protein restriction (PR) extends the lifespan and healthspan of mice. Here, we examined the effect of PR on metabolic health and the development and progression of Alzheimer's disease (AD) in the 3xTg mouse model of AD. We found that PR has metabolic benefits for 3xTg mice and non-transgenic controls of both sexes, promoting leanness and glycemic control in 3xTg mice. We found that PR induces sex-specific alterations in circulating metabolites and in the brain lipidome, downregulating sphingolipid subclasses including ceramides, glucosylceramides, and sphingomyelins in 3xTg females. Consumption of a PR diet starting at 6 months of age reduced AD pathology in conjunction with reduced mTORC1 activity, increased autophagy, and had cognitive benefits for 3xTg mice. Finally, PR improved the survival of 3xTg mice. Our results demonstrate that PR slows the progression of AD at molecular and pathological levels, preserves cognition in this mouse model of AD, and suggests that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD.

Functional role of adult hippocampal neurogenesis as a therapeutic strategy for mental disorders.

Adult neurogenesis, the process of generating new neurons from neural stem cells, plays significant roles in synaptic plasticity, memory, and mood regulation. In the mammalian brain, it continues to occur well into adulthood in discrete regions, namely, the hippocampus and olfactory bulb. During the past decade, significant progress has been made in understanding the mechanisms regulating adult hippocampal neurogenesis and its role in the etiology of mental disorders. In addition, adult hippocampal neurogenesis is highly correlated with the remission of the antidepressant effect. In this paper, we discuss three major psychiatric disorders, depression, schizophrenia, and drug addiction, in light of preclinical evidence used in establishing the neurobiological significance of adult neurogenesis. We interpret the significance of these results and pose questions that remain unanswered. Potential treatments which include electroconvulsive therapy, deep brain stimulation, chemical antidepressants, and exercise therapy are discussed. While consensus lacks on specific mechanisms, we highlight evidence which indicates that these treatments may function via an increase in neural progenitor proliferation and changes to the hippocampal circuitry. Establishing a significant role of adult neurogenesis in the pathogenicity of psychiatric disorders may hold the key to potential strategies toward effective treatment.