Apolipoprotein E ε4 modulates astrocyte neuronal support functions in the presence of amyloid-ß.

Apolipoprotein E (APOE) is a lipid transporter produced predominantly by astrocytes in the brain. The ε4 variant of APOE (APOE4) is the strongest and most common genetic risk factor for Alzheimer's disease (AD). Although the molecular mechanisms of this increased risk are unclear, APOE4 is known to alter immune signaling and lipid and glucose metabolism. Astrocytes provide various forms of support to neurons, including regulating neuronal metabolism and immune responses through cytokine signaling. Changes in astrocyte function because of APOE4 may therefore decrease neuronal support, leaving neurons more vulnerable to stress and disease insults. To determine whether APOE4 alters astrocyte neuronal support functions, we measured glycolytic and oxidative metabolism of neurons treated with conditioned media from APOE4 or APOE3 (the common, risk-neutral variant) primary astrocyte cultures. We found that APOE4 neurons treated with conditioned media from resting APOE4 astrocytes had similar metabolism to APOE3 neurons treated with media from resting APOE3 astrocytes, but treatment with astrocytic conditioned media from astrocytes challenged with amyloid-ß (Aß), a key pathological protein in AD, caused APOE4 neurons to increase their basal mitochondrial and glycolytic metabolic rates more than APOE3 neurons. These changes were not because of differences in astrocytic lactate production or glucose utilization, but instead correlated with increased glycolytic ATP production and a lack of cytokine secretion in response to Aß. Additionally, we identified that astrocytic cytokine signatures could predict basal metabolism of neurons treated with the astrocytic conditioned media. Together, these findings suggest that in the presence of Aß, APOE4 astrocytes alter immune and metabolic functions that result in a compensatory increase in neuronal metabolic stress.

Differential responses of primary neuron-secreted MCP-1 and IL-9 to type 2 diabetes and Alzheimer's disease-associated metabolites.

Type 2 diabetes (T2D) is implicated as a risk factor for Alzheimer's disease (AD), the most common form of dementia. In this work, we investigated neuroinflammatory responses of primary neurons to potentially circulating, blood-brain barrier (BBB) permeable metabolites associated with AD, T2D, or both. We identified nine metabolites associated with protective or detrimental properties of AD and T2D in literature (lauric acid, asparagine, fructose, arachidonic acid, aminoadipic acid, sorbitol, retinol, tryptophan, niacinamide) and stimulated primary mouse neuron cultures with each metabolite before quantifying cytokine secretion via Luminex. We employed unsupervised clustering, inferential statistics, and partial least squares discriminant analysis to identify relationships between cytokine concentration and disease-associations of metabolites. We identified MCP-1, a cytokine associated with monocyte recruitment, as differentially abundant between neurons stimulated by metabolites associated with protective and detrimental properties of AD and T2D. We also identified IL-9, a cytokine that promotes mast cell growth, to be differentially associated with T2D. Indeed, cytokines, such as MCP-1 and IL-9, released from neurons in response to BBB-permeable metabolites associated with T2D may contribute to AD development by downstream effects of neuroinflammation.

Predictive link between systemic metabolism and cytokine signatures in the brain of apolipoprotein E ε4 mice.

The ε4 variant of apolipoprotein E (APOE) is the strongest and most common genetic risk factor for Alzheimer's disease (AD). While the mechanism of conveyed risk is incompletely understood, promotion of inflammation, dysregulated metabolism, and protein misfolding and aggregation are contributors to accelerating disease. Here we determined the concurrent effects of systemic metabolic changes and brain inflammation in young (3-month-old) and aged (18-month-old) male and female mice carrying the APOE4 gene. Using functional metabolic assays alongside multivariate modeling of hippocampal cytokine levels, we found that brain cytokine signatures are predictive of systemic metabolic outcomes, independent of AD proteinopathies. Male and female mice each produce different cytokine signatures as they age and as their systemic metabolic phenotype declines, and these signatures are APOE genotype dependent. Ours is the first study to identify a quantitative and predictive link between systemic metabolism and specific pathological cytokine signatures in the brain. Our results highlight the effects of APOE4 beyond the brain and suggest the potential for bi-directional influence of risk factors in the brain and periphery.

Alzheimer's disease-specific cytokine secretion suppresses neuronal mitochondrial metabolism.

Introduction: Neuroinflammation and metabolic dysfunction are early alterations in Alzheimer's disease brain that are thought to contribute to disease onset and progression. Glial activation due to protein deposition results in cytokine secretion and shifts in brain metabolism, which have been observed in Alzheimer's disease patients. However, the mechanism by which this immunometabolic feedback loop can injure neurons and cause neurodegeneration remains unclear. Methods: We used Luminex XMAP technology to quantify hippocampal cytokine concentrations in the 5xFAD mouse model of Alzheimer's disease at milestone timepoints in disease development. We used partial least squares regression to build cytokine signatures predictive of disease progression, as compared to healthy aging in wild-type littermates. We applied the disease-defining cytokine signature to wild-type primary neuron cultures and measured downstream changes in gene expression using the NanoString nCounter system and mitochondrial function using the Seahorse Extracellular Flux live-cell analyzer. Results: We identified a pattern of up-regulated IFNγ, IP-10, and IL-9 as predictive of advanced disease. When healthy neurons were exposed to these cytokines in proportions found in diseased brain, gene expression of mitochondrial electron transport chain complexes, including ATP synthase, was suppressed. In live cells, basal and maximal mitochondrial respiration were impaired following cytokine stimulation. Conclusions: An Alzheimer's disease-specific pattern of cytokine secretion reduces expression of mitochondrial electron transport complexes and impairs mitochondrial respiration in healthy neurons. We establish a mechanistic link between disease-specific immune cues and impaired neuronal metabolism, potentially causing neuronal vulnerability and susceptibility to degeneration in Alzheimer's disease.

Amyloid-ß Pathology-Specific Cytokine Secretion Suppresses Neuronal Mitochondrial Metabolism.

Introduction: Neuroinflammation and metabolic dysfunction are early alterations in Alzheimer's disease (AD) brain that are thought to contribute to disease onset and progression. Glial activation due to protein deposition results in cytokine secretion and shifts in brain metabolism, which have been observed in AD patients. However, the mechanism by which this immunometabolic feedback loop can injure neurons and cause neurodegeneration remains unclear. Methods: We used Luminex XMAP technology to quantify hippocampal cytokine concentrations in the 5xFAD mouse model of AD at milestone timepoints in disease development. We used partial least squares regression to build cytokine signatures predictive of disease progression, as compared to healthy aging in wild-type littermates. We applied the disease-defining cytokine signature to wild-type primary neuron cultures and measured downstream changes in gene expression using the NanoString nCounter system and mitochondrial function using the Seahorse Extracellular Flux live-cell analyzer. Results: We identified a pattern of up-regulated IFNγ, IP-10/CXCL10, and IL-9 as predictive of advanced disease. When healthy neurons were exposed to these cytokines in proportions found in diseased brain, gene expression of mitochondrial electron transport chain complexes, including ATP synthase, was suppressed. In live cells, basal and maximal mitochondrial respiration were impaired following cytokine stimulation. Conclusions: We identify a pattern of cytokine secretion predictive of progressing amyloid-ß pathology in the 5xFAD mouse model of AD that reduces expression of mitochondrial electron transport complexes and impairs mitochondrial respiration in healthy neurons. We establish a mechanistic link between disease-specific immune cues and impaired neuronal metabolism, potentially causing neuronal vulnerability and susceptibility to degeneration in AD. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00782-y.

Monosialotetrahexosylganglioside Promotes Early Aß42 Oligomer Formation and Maintenance.

The aggregation of the amyloid beta (Aß) peptide is associated with Alzheimer's disease (AD) pathogenesis. Cell membrane composition, especially monosialotetrahexosylganglioside (GM1), is known to promote the formation of Aß fibrils, yet little is known about the roles of GM1 in the early steps of Aß oligomer formation. Here, by using GM1-contained liposomes as a mimic of the neuronal cell membrane, we demonstrate that GM1 is a critical trigger of Aß oligomerization and aggregation. We find that GM1 not only promotes the formation of Aß fibrils but also facilitates the maintenance of Aß42 oligomers on liposome membranes. We structurally characterize the Aß42 oligomers formed on the membrane and find that GM1 captures Aß by binding to its arginine-5 residue. To interrogate the mechanism of Aß42 oligomer toxicity, we design a new liposome-based Ca2+-encapsulation assay and provide new evidence for the Aß42 ion channel hypothesis. Finally, we determine the toxicity of Aß42 oligomers formed on membranes. Overall, by uncovering the roles of GM1 in mediating early Aß oligomer formation and maintenance, our work provides a novel direction for pharmaceutical research for AD.

Astrocytic Propagation of Tau in the Context of Alzheimer's Disease.

More than 6 million Americans are currently living with Alzheimer's disease (AD), and the incidence is growing rapidly with our aging population. Numerous therapeutics have failed to make it to the clinic, potentially due to a focus on presumptive pathogenic proteins instead of cell-type-specific signaling mechanisms. The tau propagation hypothesis that inter-neuronal tau transfer drives AD pathology has recently garnered attention, as accumulation of pathological tau in the brain has high clinical significance in correlating with progression of cognitive AD symptoms. However, studies on tau pathology in AD are classically neuron-centric and have greatly overlooked cell-type specific effects of tau internalization, degradation, and propagation. While the contribution of microglia to tau processing and propagation is beginning to be recognized and understood, astrocytes, glial cells in the brain important for maintaining neuronal metabolic, synaptic, trophic, and immune function which can produce, internalize, degrade, and propagate tau are understudied in their ability to affect AD progression through tau pathology. Here, we showcase evidence for whether tau uptake by astrocytes may be beneficial or detrimental to neuronal health and how astrocytes and their immunometabolic functions may be key targets for future successful AD therapies.

Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation.

Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes.

Tristetraprolin targets Nos2 expression in the colonic epithelium.

Tristetraprolin (TTP), encoded by the Zfp36 gene, is a zinc-finger protein that regulates RNA stability primarily through association with 3' untranslated regions (3' UTRs) of target mRNAs. While TTP is expressed abundantly in the intestines, its function in intestinal epithelial cells (IECs) is unknown. Here we used a cre-lox system to remove Zfp36 in the mouse epithelium to uncover a role for TTP in IECs and to identify target genes in these cells. While TTP was largely dispensable for establishment and maintenance of the colonic epithelium, we found an expansion of the proliferative zone and an increase in goblet cell numbers in the colon crypts of Zfp36ΔIEC mice. Furthermore, through RNA-sequencing of transcripts isolated from the colons of Zfp36fl/fl and Zfp36ΔIEC mice, we found that expression of inducible nitric oxide synthase (iNos or Nos2) was elevated in TTP-knockout IECs. We demonstrate that TTP interacts with AU-rich elements in the Nos2 3' UTR and suppresses Nos2 expression. In comparison to control Zfp36fl/fl mice, Zfp36ΔIEC mice were less susceptible to dextran sodium sulfate (DSS)-induced acute colitis. Together, these results demonstrate that TTP in IECs targets Nos2 expression and aggravates acute colitis.