Microglial mTOR Activation Upregulates Trem2 and Enhances ß-Amyloid Plaque Clearance in the 5XFAD Alzheimer's Disease Model.

The mechanistic target of rapamycin (mTOR) signaling pathway plays a major role in key cellular processes including metabolism and differentiation; however, the role of mTOR in microglia and its importance in Alzheimer's disease (AD) have remained largely uncharacterized. We report that selective loss of Tsc1, a negative regulator of mTOR, in microglia in mice of both sexes, caused mTOR activation and upregulation of Trem2 with enhanced ß-Amyloid (Aß) clearance, reduced spine loss, and improved cognitive function in the 5XFAD AD mouse model. Combined loss of Tsc1 and Trem2 in microglia led to reduced Aß clearance and increased Aß plaque burden revealing that Trem2 functions downstream of mTOR. Tsc1 mutant microglia showed increased phagocytosis with upregulation of CD68 and Lamp1 lysosomal proteins. In vitro studies using Tsc1-deficient microglia revealed enhanced endocytosis of the lysosomal tracker indicator Green DND-26 suggesting increased lysosomal activity. Incubation of Tsc1-deficient microglia with fluorescent-labeled Aß revealed enhanced Aß uptake and clearance, which was blunted by rapamycin, an mTOR inhibitor. In vivo treatment of mice of relevant genotypes in the 5XFAD background with rapamycin, affected microglial activity, decreased Trem2 expression and reduced Aß clearance causing an increase in Aß plaque burden. Prolonged treatment with rapamycin caused even further reduction of mTOR activity, reduction in Trem2 expression, and increase in Aß levels. Together, our findings reveal that mTOR signaling in microglia is critically linked to Trem2 regulation and lysosomal biogenesis, and that the upregulation of Trem2 in microglia through mTOR activation could be exploited toward better therapeutic avenues to Aß-related AD pathologies.SIGNIFICANCE STATEMENT Mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator for major cellular metabolic processes. However, the link between mTOR signaling and Alzheimer's disease (AD) is not well understood. In this study, we provide compelling in vivo evidence that mTOR activation in microglia would benefit ß-Amyloid (Aß)-related AD pathologies, as it upregulates Trem2, a key receptor for Aß plaque uptake. Inhibition of mTOR pathway with rapamycin, a well-established immunosuppressant, downregulated Trem2 in microglia and reduced Aß plaque clearance indicating that mTOR inactivation may be detrimental in Aß-associated AD patients. This finding will have a significant public health impact and benefit, regarding the usage of rapamycin in AD patients, which we believe will aggravate the Aß-related AD pathologies.

Interactive Effects of Empagliflozin and Hyperglycemia on Urinary Amino Acids in Individuals With Type 1 Diabetes.

Optimizing energy use in the kidney is critical for normal kidney function. Here, we investigate the effect of hyperglycemia and sodium-glucose cotransporter 2 (SGLT2) inhibition on urinary amino acid excretion in individuals with type 1 diabetes (T1D). The open-label ATIRMA trial assessed the impact of 8 weeks of 25 mg empagliflozin orally once per day in 40 normotensive normoalbuminuric young adults with T1D. A consecutive 2-day assessment of clamped euglycemia and hyperglycemia was evaluated at baseline and posttreatment visits. Principal component analysis was performed on urinary amino acids grouped into representative metabolic pathways using MetaboAnalyst. At baseline, acute hyperglycemia was associated with changes in 25 of the 33 urinary amino acids or their metabolites. The most significant amino acid metabolites affected by acute hyperglycemia were 3-hydroxykynurenine, serotonin, glycyl-histidine, and nicotinic acid. The changes in amino acid metabolites were reflected by the induction of four biosynthetic pathways: aminoacyl-tRNA; valine, leucine, and isoleucine; arginine; and phenylalanine, tyrosine, and tryptophan. In acute hyperglycemia, empagliflozin significantly attenuated the increases in aminoacyl-tRNA biosynthesis and valine, leucine, and isoleucine biosynthesis. Our findings using amino acid metabolomics indicate that hyperglycemia stimulates biosynthetic pathways in T1D. SGLT2 inhibition may attenuate the increase in biosynthetic pathways to optimize kidney energy metabolism.

Female Protection Against Diabetic Kidney Disease Is Regulated by Kidney-Specific AMPK Activity.

Reduced kidney AMPK activity is associated with nutrient stress-induced chronic kidney disease (CKD) in male mice. In contrast, female mice resist nutrient stress-induced CKD. The role of kidney AMPK in sex-related organ protection against nutrient stress and metabolite changes was evaluated in diabetic kidney tubule-specific AMPKγ2KO (KTAMPKγ2ΚΟ) male and female mice. In wild-type (WT) males, diabetes increased albuminuria, urinary kidney injury molecule-1, hypertension, kidney p70S6K phosphorylation, and kidney matrix accumulation; these features were not exacerbated with KTAMPKγ2ΚΟ. Whereas WT females had protection against diabetes-induced kidney injury, KTAMPKγ2ΚΟ led to loss of female protection against kidney disease. The hormone 17ß-estradiol ameliorated high glucose-induced AMPK inactivation, p70S6K phosphorylation, and matrix protein accumulation in kidney tubule cells. The mechanism for female protection against diabetes-induced kidney injury is likely via an estrogen-AMPK pathway, as inhibition of AMPK led to loss of estrogen protection to glucose-induced mTORC1 activation and matrix production. RNA sequencing and metabolomic analysis identified a decrease in the degradation pathway of phenylalanine and tyrosine resulting in increased urinary phenylalanine and tyrosine levels in females. The metabolite levels correlated with loss of female protection. The findings provide new insights to explain evolutionary advantages to females during states of nutrient challenges.

Quinolinic acid links kidney injury to brain toxicity.

Kidney dysfunction often leads to neurological impairment, yet the complex kidney-brain relationship remains elusive. We employed spatial and bulk metabolomics to investigate a mouse model of rapid kidney failure induced by mouse double minute 2 ( Mdm2) conditional deletion in the kidney tubules to interrogate kidney and brain metabolism. Pathway enrichment analysis of focused plasma metabolomics panel pinpointed tryptophan metabolism as the most altered pathway with kidney failure. Spatial metabolomics showed toxic tryptophan metabolites in the kidneys and brains, revealing a novel connection between advanced kidney disease and accelerated kynurenine degradation. In particular, the excitotoxic metabolite quinolinic acid was localized in ependymal cells adjacent to the ventricle in the setting of kidney failure. These findings were associated with brain inflammation and cell death. A separate mouse model of acute kidney injury also had an increase in circulating toxic tryptophan metabolites along with altered brain inflammation. Patients with advanced CKD similarly demonstrated elevated plasma kynurenine metabolites and quinolinic acid was uniquely correlated with fatigue and reduced quality of life in humans. Overall, our study identifies the kynurenine pathway as a bridge between kidney decline, systemic inflammation, and brain toxicity, offering potential avenues for diagnosis and treatment of neurological issues in kidney disease.

Ketogenic diet induces p53-dependent cellular senescence in multiple organs.

A ketogenic diet (KD) is a high-fat, low-carbohydrate diet that leads to the generation of ketones. While KDs improve certain health conditions and are popular for weight loss, detrimental effects have also been reported. Here, we show mice on two different KDs and, at different ages, induce cellular senescence in multiple organs, including the heart and kidney. This effect is mediated through adenosine monophosphate-activated protein kinase (AMPK) and inactivation of mouse double minute 2 (MDM2) by caspase-2, leading to p53 accumulation and p21 induction. This was established using p53 and caspase-2 knockout mice and inhibitors to AMPK, p21, and caspase-2. In addition, senescence-associated secretory phenotype biomarkers were elevated in serum from mice on a KD and in plasma samples from patients on a KD clinical trial. Cellular senescence was eliminated by a senolytic and prevented by an intermittent KD. These results have important clinical implications, suggesting that the effects of a KD are contextual and likely require individual optimization.

Evaluation of Exploratory Fluid Biomarker Results from a Phase 1 Senolytic Trial in Mild Alzheimer's Disease.

Senescent cell accumulation contributes to the progression of age-related disorders including Alzheimer's disease (AD). Clinical trials evaluating senolytics, drugs that clear senescent cells, are underway, but lack standardized outcome measures. Our team recently published data from the first open-label trial to evaluate senolytics (dasatinib plus quercetin) in AD. After 12-weeks of intermittent treatment, we reported brain exposure to dasatinib, favorable safety and tolerability, and modest post-treatment changes in cerebrospinal fluid (CSF) inflammatory and AD biomarkers using commercially available assays. Herein, we present more comprehensive exploratory analyses of senolytic associated changes in AD relevant proteins, metabolites, lipids, and transcripts measured across blood, CSF, and urine. These analyses included mass spectrometry for precise quantification of amyloid beta (Aß) and tau in CSF; immunoassays to assess senescence associated secretory factors in plasma, CSF, and urine; mass spectrometry analysis of urinary metabolites and lipids in blood and CSF; and transcriptomic analyses relevant to chronic stress measured in peripheral blood cells. Levels of Aß and tau species remained stable. Targeted cytokine and chemokine analyses revealed treatment-associated increases in inflammatory plasma fractalkine and MMP-7 and CSF IL-6. Urinary metabolites remained unchanged. Modest treatment-associated lipid profile changes suggestive of decreased inflammation were observed both peripherally and centrally. Blood transcriptomic analysis indicated downregulation of inflammatory genes including FOS, FOSB, IL1ß, IL8, JUN, JUNB, PTGS2. These data provide a foundation for developing standardized outcome measures across senolytic studies and indicate distinct biofluid-specific signatures that will require validation in future studies. ClinicalTrials.gov: NCT04063124.

Glycolytic lactate in diabetic kidney disease.

Lactate elevation is a well-characterized biomarker of mitochondrial dysfunction, but its role in diabetic kidney disease (DKD) is not well defined. Urine lactate was measured in patients with type 2 diabetes (T2D) in 3 cohorts (HUNT3, SMART2D, CRIC). Urine and plasma lactate were measured during euglycemic and hyperglycemic clamps in participants with type 1 diabetes (T1D). Patients in the HUNT3 cohort with DKD had elevated urine lactate levels compared with age- and sex-matched controls. In patients in the SMART2D and CRIC cohorts, the third tertile of urine lactate/creatinine was associated with more rapid estimated glomerular filtration rate decline, relative to first tertile. Patients with T1D demonstrated a strong association between glucose and lactate in both plasma and urine. Glucose-stimulated lactate likely derives in part from proximal tubular cells, since lactate production was attenuated with sodium-glucose cotransporter-2 (SGLT2) inhibition in kidney sections and in SGLT2-deficient mice. Several glycolytic genes were elevated in human diabetic proximal tubules. Lactate levels above 2.5 mM potently inhibited mitochondrial oxidative phosphorylation in human proximal tubule (HK2) cells. We conclude that increased lactate production under diabetic conditions can contribute to mitochondrial dysfunction and become a feed-forward component to DKD pathogenesis.

Crabtree effect in kidney proximal tubule cells via late-stage glycolytic intermediates.

The Crabtree effect is defined as a rapid glucose-induced repression of mitochondrial oxidative metabolism and has been described in yeasts and tumor cells. Using plate-based respirometry, we identified the Crabtree effect in normal (non-tumor) kidney proximal tubule epithelial cells (PTEC) but not in other kidney cells (podocytes or mesangial cells) or mammalian cells (C2C12 myoblasts). Glucose-induced repression of respiration was prevented by reducing glycolysis at the proximal step with 2-deoxyglucose and partially reversed by pyruvate. The late-stage glycolytic intermediates glyceraldehyde 3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate, but not the early-stage glycolytic intermediates or lactate, inhibited respiration in permeabilized PTEC and kidney cortex mitochondria, mimicking the Crabtree effect. Studies in diabetic mice indicated a pattern of increased late-stage glycolytic intermediates consistent with a similar pattern occurring in vivo. Our results show the unique presence of the Crabtree effect in kidney PTEC and identify the major mediators of this effect.

Functional Characterization of TMEM127 Variants Reveals Novel Insights into Its Membrane Topology and Trafficking.

CONTEXT: TMEM127 is a poorly known tumor suppressor gene associated with pheochromocytomas, paragangliomas, and renal carcinomas. Our incomplete understanding of TMEM127 function has limited our ability to predict variant pathogenicity. PURPOSE: To better understand the function of the transmembrane protein TMEM127 we undertook cellular and molecular evaluation of patient-derived germline variants. DESIGN: Subcellular localization and steady-state levels of tumor-associated, transiently expressed TMEM127 variants were compared to the wild-type protein using immunofluorescence and immunoblot analysis, respectively, in cells genetically modified to lack endogenous TMEM127. Membrane topology and endocytic mechanisms were also assessed. RESULTS: We identified 3 subgroups of mutations and determined that 71% of the variants studied are pathogenic or likely pathogenic through loss of membrane-binding ability, stability, and/or internalization capability. Investigation into an N-terminal cluster of missense variants uncovered a previously unrecognized transmembrane domain, indicating that TMEM127 is a 4- transmembrane, not a 3-transmembrane domain-containing protein. Additionally, a C-terminal variant with predominant plasma membrane localization revealed an atypical, extended acidic, dileucine-based motif required for TMEM127 internalization through clathrin-mediated endocytosis. CONCLUSION: We characterized the functional deficits of several germline TMEM127 variants and identified novel structure-function features of TMEM127. These findings will assist in determining pathogenicity of TMEM127 variants and will help guide future studies investigating the cellular role of TMEM127.

Partial loss of CFIm25 causes learning deficits and aberrant neuronal alternative polyadenylation.

We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (Gennarino et al., 2015). However, the patients' CNVs also included other genes. To determine if reduced NUDT21 function alone can cause disease, we generated Nudt21+/- mice to mimic NUDT21-deletion patients. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits, cortical hyperexcitability, and misregulated alternative polyadenylation (APA) in their hippocampi. Further, to determine the mediators driving neural dysfunction in humans, we partially inhibited NUDT21 in human stem cell-derived neurons to reduce CFIm25 by 30%. This induced APA and protein level misregulation in hundreds of genes, a number of which cause intellectual disability when mutated. Altogether, these results show that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.

Tubulin Polymerization Promoting Protein, Ringmaker, and MAP1B Homolog Futsch Coordinate Microtubule Organization and Synaptic Growth.

Drosophila Ringmaker (Ringer) is homologous to the human Tubulin Polymerization Promoting Proteins (TPPPs) that are implicated in the stabilization and bundling of microtubules (MTs) that are particularly important for neurons and are also implicated in synaptic organization and plasticity. No in vivo functional data exist that have addressed the role of TPPP in synapse organization in any system. Here, we present the phenotypic and functional characterization of ringer mutants during Drosophila larval neuromuscular junction (NMJ) synaptic development. ringer mutants show reduced synaptic growth and transmission and display phenotypic similarities and genetic interactions with the Drosophila homolog of vertebrate Microtubule Associated Protein (MAP)1B, futsch. Immunohistochemical and biochemical analyses show that individual and combined loss of Ringer and Futsch cause a significant reduction in MT loops at the NMJs and reduced acetylated-tubulin levels. Presynaptic over-expression of Ringer and Futsch causes elevated levels of acetylated-tubulin and significant increase in NMJ MT loops. These results indicate that Ringer and Futsch regulate synaptic MT organization in addition to synaptic growth. Together our findings may inform studies on the close mammalian homolog, TPPP, and provide insights into the role of MTs and associated proteins in synapse growth and organization.

Role of the Retinoblastoma protein, Rb, during adult neurogenesis in the olfactory bulb.

Adult neural stem cells (aNSCs) are relatively quiescent populations that give rise to distinct neuronal subtypes throughout life, yet, at a very low rate and restricted differentiation potential. Thus, identifying the molecular mechanisms that control their cellular expansion is critical for regeneration after brain injury. Loss of the Retinoblastoma protein, Rb, leads to several defects in cell cycle as well as neuronal differentiation and migration during brain development. Here, we investigated the role of Rb during adult neurogenesis in the olfactory bulb (OB) by inducing its temporal deletion in aNSCs and progenitors. Loss of Rb was associated with increased proliferation of adult progenitors in the subventricular zone (SVZ) and the rostral migratory stream (RMS) but did not alter self-renewal of aNSCs or neuroblasts subsequent migration and terminal differentiation. Hence, one month after their birth, Rb-null neuroblasts were able to differentiate into distinct subtypes of GABAergic OB interneurons but were gradually lost after 3 months. Similarly, Rb controlled aNSCs/progenitors proliferation in vitro without affecting their differentiation capacity. This enhanced SVZ/OB neurogenesis associated with loss of Rb was only transient and negatively affected by increased apoptosis indicating a critical requirement for Rb in the long-term survival of adult-born OB interneurons.