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.

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.

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.

Mitochondrial regulator PGC-1a in neuronal metabolism and brain aging.

The brain is a high energy tissue, and the cell types of which it is comprised are distinct in function and in metabolic requirements. The transcriptional co-activator PGC-1a is a master regulator of mitochondrial function and is highly expressed in the brain; however, its cell-type specific role in regulating metabolism has not been well established. Here, we show that PGC-1a is responsive to aging and that expression of the neuron specific PGC-1a isoform allows for specialization in metabolic adaptation. Transcriptional profiles of the cortex from male mice show an impact of age on immune, inflammatory, and neuronal functional pathways and a highly integrated metabolic response that is associated with decreased expression of PGC-1a. Proteomic analysis confirms age-related changes in metabolism and further shows changes in ribosomal and RNA splicing pathways. We show that neurons express a specialized PGC-1a isoform that becomes active during differentiation from stem cells and is further induced during the maturation of isolated neurons. Neuronal but not astrocyte PGC-1a responds robustly to inhibition of the growth sensitive kinase GSK3b, where the brain specific promoter driven dominant isoform is repressed. The GSK3b inhibitor lithium broadly reprograms metabolism and growth signaling, including significantly lower expression of mitochondrial and ribosomal pathway genes and suppression of growth signaling, which are linked to changes in mitochondrial function and neuronal outgrowth. In vivo, lithium treatment significantly changes the expression of genes involved in cortical growth, endocrine, and circadian pathways. These data place the GSK3b/PGC-1a axis centrally in a growth and metabolism network that is directly relevant to brain aging.

The citrate transporters SLC13A5 and SLC25A1 elicit different metabolic responses and phenotypes in the mouse.

Cytosolic citrate is imported from the mitochondria by SLC25A1, and from the extracellular milieu by SLC13A5. In the cytosol, citrate is used by ACLY to generate acetyl-CoA, which can then be exported to the endoplasmic reticulum (ER) by SLC33A1. Here, we report the generation of mice with systemic overexpression (sTg) of SLC25A1 or SLC13A5. Both animals displayed increased cytosolic levels of citrate and acetyl-CoA; however, SLC13A5 sTg mice developed a progeria-like phenotype with premature death, while SLC25A1 sTg mice did not. Analysis of the metabolic profile revealed widespread differences. Furthermore, SLC13A5 sTg mice displayed increased engagement of the ER acetylation machinery through SLC33A1, while SLC25A1 sTg mice did not. In conclusion, our findings point to different biological responses to SLC13A5- or SLC25A1-mediated import of citrate and suggest that the directionality of the citrate/acetyl-CoA pathway can transduce different signals.

Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging.

Single-cell (SC) analysis provides unique insight into individual cell dynamics and cell-to-cell heterogeneity. Here, we utilize trapped ion mobility separation coupled with dual-polarity ionization mass spectrometry imaging (MSI) to enable high-throughput in situ profiling of the SC lipidome. Multimodal SC imaging, in which dual-polarity-mode MSI is used to perform serial data acquisition runs on individual cells, significantly enhanced SC lipidome coverage. High-spatial resolution SC-MSI identifies both inter- and intracellular lipid heterogeneity; this heterogeneity is further explicated by Uniform Manifold Approximation and Projection and machine learning-driven classifications. We characterize SC lipidome alteration in response to stearoyl-CoA desaturase 1 inhibition and, additionally, identify cell-layer specific lipid distribution patterns in mouse cerebellar cortex. This integrated multimodal SC-MSI technology enables high-resolution spatial mapping of intercellular and cell-to-cell lipidome heterogeneity, SC lipidome remodeling induced by pharmacological intervention, and region-specific lipid diversity within tissue.

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.

Defective PTEN-induced kinase 1/Parkin mediated mitophagy and neurodegenerative diseases.

The selective degradation of mitochondria through mitophagy is a crucial process for maintaining mitochondrial function and cellular health. Mitophagy is a specialized form of selective autophagy that uses unique machinery to recognize and target damaged mitochondria for mitophagosome- and lysosome-dependent degradation. This process is particularly important in cells with high metabolic activity like neurons, and the accumulation of defective mitochondria is a common feature among neurodegenerative disorders. Here, we describe essential steps involved in the induction and progression of mitophagy, and then highlight the various mechanisms that specifically contribute to defective mitophagy in highly prevalent neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and Amyotrophic Lateral Sclerosis.

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.

ATase inhibition rescues age-associated proteotoxicity of the secretory pathway.

Malfunction of autophagy contributes to the progression of many chronic age-associated diseases. As such, improving normal proteostatic mechanisms is an active target for biomedical research and a key focal area for aging research. Endoplasmic reticulum (ER)-based acetylation has emerged as a mechanism that ensures proteostasis within the ER by regulating the induction of ER specific autophagy. ER acetylation is ensured by two ER-membrane bound acetyltransferases, ATase1 and ATase2. Here, we show that ATase inhibitors can rescue ongoing disease manifestations associated with the segmental progeria-like phenotype of AT-1 sTg mice. We also describe a pipeline to reliably identify ATase inhibitors with promising druggability properties. Finally, we show that successful ATase inhibitors can rescue the proteopathy of a mouse model of Alzheimer's disease. In conclusion, our study proposes that ATase-targeting approaches might offer a translational pathway for many age-associated proteopathies affecting the ER/secretory pathway.

SLC13A5/sodium-citrate co-transporter overexpression causes disrupted white matter integrity and an autistic-like phenotype.

Endoplasmic reticulum-based N ɛ-lysine acetylation serves as an important protein quality control system for the secretory pathway. Dysfunctional endoplasmic reticulum-based acetylation, as caused by overexpression of the acetyl coenzyme A transporter AT-1 in the mouse, results in altered glycoprotein flux through the secretory pathway and an autistic-like phenotype. AT-1 works in concert with SLC25A1, the citrate/malate antiporter in the mitochondria, SLC13A5, the plasma membrane sodium/citrate symporter and ATP citrate lyase, the cytosolic enzyme that converts citrate into acetyl coenzyme A. Here, we report that mice with neuron-specific overexpression of SLC13A5 exhibit autistic-like behaviours with a jumping stereotypy. The mice displayed disrupted white matter integrity and altered synaptic structure and function. Analysis of both the proteome and acetyl-proteome revealed unique adaptations in the hippocampus and cortex, highlighting a metabolic response that likely plays an important role in the SLC13A5 neuron transgenic phenotype. Overall, our results support a mechanistic link between aberrant intracellular citrate/acetyl coenzyme A flux and the development of an autistic-like phenotype.

Endoplasmic reticulum acetyltransferases Atase1 and Atase2 differentially regulate reticulophagy, macroautophagy and cellular acetyl-CoA metabolism.

Nε-lysine acetylation in the ER lumen is a recently discovered quality control mechanism that ensures proteostasis within the secretory pathway. The acetyltransferase reaction is carried out by two type-II membrane proteins, ATase1/NAT8B and ATase2/NAT8. Prior studies have shown that reducing ER acetylation can induce reticulophagy, increase ER turnover, and alleviate proteotoxic states. Here, we report the generation of Atase1-/- and Atase2-/- mice and show that these two ER-based acetyltransferases play different roles in the regulation of reticulophagy and macroautophagy. Importantly, knockout of Atase1 alone results in activation of reticulophagy and rescue of the proteotoxic state associated with Alzheimer's disease. Furthermore, loss of Atase1 or Atase2 results in widespread adaptive changes in the cell acetylome and acetyl-CoA metabolism. Overall, our study supports a divergent role of Atase1 and Atase2 in cellular biology, emphasizing ATase1 as a valid translational target for diseases characterized by toxic protein aggregation in the secretory pathway.

ATG9A regulates proteostasis through reticulophagy receptors FAM134B and SEC62 and folding chaperones CALR and HSPB1.

The acetylation of ATG9A within the endoplasmic reticulum (ER) lumen regulates the induction of reticulophagy. ER acetylation is ensured by AT-1/SLC33A1, a membrane transporter that maintains the cytosol-to-ER flux of acetyl-CoA. Defective AT-1 activity, as caused by heterozygous/homozygous mutations and gene duplication events, results in severe disease phenotypes. Here, we show that although the acetylation of ATG9A occurs in the ER lumen, the induction of reticulophagy requires ATG9A to engage FAM134B and SEC62 on the cytosolic side of the ER. To address this conundrum, we resolved the ATG9A interactome in two mouse models of AT-1 dysregulation: AT-1 sTg, a model of systemic AT-1 overexpression with hyperacetylation of ATG9A, and AT-1S113R/+, a model of AT-1 haploinsufficiency with hypoacetylation of ATG9A. We identified CALR and HSPB1 as two ATG9A partners that regulate the induction of reticulophagy as a function of ATG9A acetylation and discovered that ATG9A associates with several proteins that maintain ER proteostasis.

Acetyl-CoA flux from the cytosol to the ER regulates engagement and quality of the secretory pathway.

Nε-lysine acetylation in the ER is an essential component of the quality control machinery. ER acetylation is ensured by a membrane transporter, AT-1/SLC33A1, which translocates cytosolic acetyl-CoA into the ER lumen, and two acetyltransferases, ATase1 and ATase2, which acetylate nascent polypeptides within the ER lumen. Dysfunctional AT-1, as caused by gene mutation or duplication events, results in severe disease phenotypes. Here, we used two models of AT-1 dysregulation to investigate dynamics of the secretory pathway: AT-1 sTg, a model of systemic AT-1 overexpression, and AT-1S113R/+, a model of AT-1 haploinsufficiency. The animals displayed reorganization of the ER, ERGIC, and Golgi apparatus. In particular, AT-1 sTg animals displayed a marked delay in Golgi-to-plasma membrane protein trafficking, significant alterations in Golgi-based N-glycan modification, and a marked expansion of the lysosomal network. Collectively our results indicate that AT-1 is essential to maintain proper organization and engagement of the secretory pathway.

Deficient Endoplasmic Reticulum Acetyl-CoA Import in Pancreatic Acinar Cells Leads to Chronic Pancreatitis.

BACKGROUND & AIMS: Maintaining endoplasmic reticulum (ER) proteostasis is essential for pancreatic acinar cell function. Under conditions of severe ER stress, activation of pathogenic unfolded protein response pathways plays a central role in the development and progression of pancreatitis. Less is known, however, of the consequence of perturbing ER-associated post-translational protein modifications on pancreatic outcomes. Here, we examined the role of the ER acetyl-CoA transporter AT-1 on pancreatic homeostasis. METHODS: We used an AT-1S113R/+ hypomorphic mouse model, and generated an inducible, acinar-specific, AT-1 knockout mouse model, and performed histologic and biochemical analyses to probe the effect of AT-1 loss on acinar cell physiology. RESULTS: We found that AT-1 expression is down-regulated significantly during both acute and chronic pancreatitis. Furthermore, acinar-specific deletion of AT-1 in acinar cells induces chronic ER stress marked by activation of both the spliced x-box binding protein 1 and protein kinase R-like ER kinase pathways, leading to spontaneous mild/moderate chronic pancreatitis evidenced by accumulation of intracellular trypsin, immune cell infiltration, and fibrosis. Induction of acute-on-chronic pancreatitis in the AT-1 model led to acinar cell loss and glad atrophy. CONCLUSIONS: These results indicate a key role for AT-1 in pancreatic acinar cell homeostasis, the unfolded protein response, and that perturbations in AT-1 function leads to pancreatic disease.

Glial Cell-Axonal Growth Cone Interactions in Neurodevelopment and Regeneration.

The developing nervous system is a complex yet organized system of neurons, glial support cells, and extracellular matrix that arranges into an elegant, highly structured network. The extracellular and intracellular events that guide axons to their target locations have been well characterized in many regions of the developing nervous system. However, despite extensive work, we have a poor understanding of how axonal growth cones interact with surrounding glial cells to regulate network assembly. Glia-to-growth cone communication is either direct through cellular contacts or indirect through modulation of the local microenvironment via the secretion of factors or signaling molecules. Microglia, oligodendrocytes, astrocytes, Schwann cells, neural progenitor cells, and olfactory ensheathing cells have all been demonstrated to directly impact axon growth and guidance. Expanding our understanding of how different glial cell types directly interact with growing axons throughout neurodevelopment will inform basic and clinical neuroscientists. For example, identifying the key cellular players beyond the axonal growth cone itself may provide translational clues to develop therapeutic interventions to modulate neuron growth during development or regeneration following injury. This review will provide an overview of the current knowledge about glial involvement in development of the nervous system, specifically focusing on how glia directly interact with growing and maturing axons to influence neuronal connectivity. This focus will be applied to the clinically-relevant field of regeneration following spinal cord injury, highlighting how a better understanding of the roles of glia in neurodevelopment can inform strategies to improve axon regeneration after injury.

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.

Mass Spectrometry Imaging of N-Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using a Novel Subatmospheric Pressure Ionization Source.

N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.

Acetyl-CoA flux regulates the proteome and acetyl-proteome to maintain intracellular metabolic crosstalk.

AT-1/SLC33A1 is a key member of the endoplasmic reticulum (ER) acetylation machinery, transporting acetyl-CoA from the cytosol into the ER lumen where acetyl-CoA serves as the acetyl-group donor for Nε-lysine acetylation. Dysfunctional ER acetylation, as caused by heterozygous or homozygous mutations as well as gene duplication events of AT-1/SLC33A1, has been linked to both developmental and degenerative diseases. Here, we investigate two models of AT-1 dysregulation and altered acetyl-CoA flux: AT-1S113R/+ mice, a model of AT-1 haploinsufficiency, and AT-1 sTg mice, a model of AT-1 overexpression. The animals display distinct metabolic adaptation across intracellular compartments, including reprogramming of lipid metabolism and mitochondria bioenergetics. Mechanistically, the perturbations to AT-1-dependent acetyl-CoA flux result in global and specific changes in both the proteome and the acetyl-proteome (protein acetylation). Collectively, our results suggest that AT-1 acts as an important metabolic regulator that maintains acetyl-CoA homeostasis by promoting functional crosstalk between different intracellular organelles.