SummaryAttenuated auto-lysosomal system has been associated with Alzheimer disease (AD), yet all underlying molecular mechanisms leading to this impairment are unknown. We show that the amino acid sensing of mechanistic target of rapamycin complex 1 (mTORC1) is dysregulated in cells deficient in presenilin, a protein associated with AD. In these cells, mTORC1 is constitutively tethered to lysosomal membranes, unresponsive to starvation, and inhibitory to TFEB-mediated clearance due to a reduction in Sestrin2 expression. Normalization of Sestrin2 levels through overexpression or elevation of nuclear calcium rescued mTORC1 tethering and initiated clearance. While CLEAR network attenuation in vivo results in buildup of amyloid, phospho-Tau, and neurodegeneration, presenilin-knockout fibroblasts and iPSC-derived AD human neurons fail to effectively initiate autophagy. These results propose an altered mechanism for nutrient sensing in presenilin deficiency and underline an importance of clearance pathways in the onset of AD.

The mechanistic target of rapamycin kinase complex 1 (MTORC1) is a central cellular kinase that integrates major signaling pathways, allowing for regulation of anabolic and catabolic processes including macroautophagy/autophagy and lysosomal biogenesis. Essential to these processes is the regulatory activity of TFEB (transcription factor EB). In a regulatory feedback loop modulating transcriptional levels of RRAG/Rag GTPases, TFEB controls MTORC1 tethering to membranes and induction of anabolic processes upon nutrient replenishment. We now show that TFEB promotes expression of endocytic genes and increases rates of cellular endocytosis during homeostatic baseline and starvation conditions. TFEB-mediated endocytosis drives assembly of the MTORC1-containing nutrient sensing complex through the formation of endosomes that carry the associated proteins RRAGD, the amino acid transporter SLC38A9, and activate AKT/protein kinase B (AKT p-T308). TFEB-induced signaling endosomes en route to lysosomes are induced by amino acid starvation and are required to dissociate TSC2, re-tether and activate MTORC1 on endolysosomal membranes. This study characterizes TFEB-mediated endocytosis as a critical process leading to activation of MTORC1 and autophagic function, thus identifying the importance of the dynamic endolysosomal system in cellular clearance. Abbreviations: CAD: central adrenergic tyrosine hydroxylase-expressing-a-differentiated; ChIP-seq: chromosome immunoprecipitation sequencing; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; EEA1: early endosomal antigen 1; EGF: epidermal growth factor; FBS: fetal bovine serum; GFP: green fluorescent protein; GTPase: guanosine triphosphatase; HEK293T: human embryonic kidney 293 cells expressing a temperature-sensitive mutant of the SV40 large T antigen; LAMP: lysosomal-associated membrane protein; LYNUS: lysosomal nutrient-sensing complex; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha/beta; MTOR: mechanistic target of rapamycin kinase; MTORC: mechanistic target of rapamycin kinase complex; OE: overexpression; PH: pleckstrin homology; PtdIns(3,4,5)P: phosphatidylinositol 3,4,5-trisphosphate; RRAGD: Ras related GTPase binding D; RHEB: Ras homolog enriched in brain; SLC38A9: solute carrier family 38 member 9; SQSTM1: sequestosome 1; TFEB: transcription factor EB; TSC2: tuberous sclerosis 2; TMR: tetramethylrhodamine; ULK1: unc-51 like kinase 1; WT: wild type.

Iron is an essential trace element required for important brain functions including oxidative metabolism, synaptic plasticity, myelination, and the synthesis of neurotransmitters. Disruptions in brain iron homeostasis underlie many neurodegenerative diseases. Increasing evidence suggests that accumulation of brain iron and chronic neuroinflammation, characterized by microglia activation and secretion of proinflammatory cytokines, are hallmarks of neurodegenerative disorders including Alzheimer’ s disease. While substantial efforts have led to an increased understanding of iron metabolism and the role of microglial cells in neuroinflammation, important questions still remain unanswered. Whether or not increased brain iron augments the inflammatory responses of microglial cells, including the molecular cues that guide such responses, is still unclear. How these brain macrophages accumulate, store, and utilize intracellular iron to carry out their various functions under normal and disease conditions is incompletely understood. Here, we describe the known and emerging mechanisms involved in microglial cell iron transport and metabolism as well as inflammatory responses in the brain, with a focus on AD.

Neonatal encephalopathy due to hypoxic-ischemic (HI) brain injury triggers a wave of neuroinflammatory events attributed to causing the progressive degeneration and functional deficits seen weeks after the initial insult. In a recent set of studies, we evaluated the therapeutic efficacy of a small molecule antagonist for ALK5 (activin-like kinase 5 ), TGF-β receptor in a rat model of moderate perinatal HI and found significant improvements in neurologic outcomes. Here, we have extended those studies to evaluate the efficacy of delayed TGF-β receptor antagonism on postnatal day (P) 6 and P9 HI rat pups with and without hypothermia. The ALK5 receptor antagonist SB505124 was administered systemically by osmotic pump beginning 3 days following HI. Extending our earlier data set that showed protection of the hippocampus in P6 pups treated with SB505124, these animals sustained less damage to their hippocampi and had improved performance on the Morris water maze (MWM) when tested on P60 versus vehicle-treated HI animals. By contrast, SB505124 did not improve sensorimotor deficits and exacerbated hippocampal and thalamic volume loss when administered 3 days after HI to P9 pups. SB505124-treated rats injured on P9 tended to perform worse than their vehicle-treated counterparts on MWM, and SB505124 treatment did not preserve hippocampal or thalamic neurons in P9 pups when combined with hypothermia. To elucidate the mechanism whereby ALK5 inhibition reduced neuronal death in the P6 HI model, we assessed levels of autophagy markers in neurons of the neocortex, hippocampus, and thalamus, and in the subcortical white matter, and found that SB505124 increased numbers of autophagosomes and levels of lipidated LC3 (light chain 3), a key protein known to mediate autophagy. Altogether, our results demonstrate that there is a dynamic switch in the CNS response to TGF-β₁ that occurs around P9 in rats where TGF-β signaling inhibition worsens functional outcomes. This response is similar to the outcome of antagonizing TGF-β signaling in adult stroke and other CNS disease models. We conclude that attenuating TGF-β₁ signaling will likely be an effective treatment for HI-related encephalopathy in moderately preterm infants, offering protection of the neocortex, hippocampus, and thalamus with enhanced cerebral autophagy contributing to the decrease in the extent of progressive neuronal cell death.

Alzheimer’s disease (AD) is characterized by accumulation of amyloid‐beta (Aβ) senile plaques in patients’ brain tissues. Elevated levels of interleukin‐1beta (IL‐1β) have been identified in cerebrospinal fluid of living AD patients and in animal models of AD. Increased expression of IL‐1β and iron accumulation have been identified in microglial cells that cluster around amyloid plaques in AD mouse models and post‐mortem brain tissues of AD patients. The goals of this study were to determine the effects of Aβ on the secretion of IL‐1β by microglial cells and whether iron status influences this pro‐inflammatory signaling cue. Immortalized microglial (IMG) cells were incubated with Aβ with or without iron. qRT‐PCR and western blot analyses showed that Aβ induces biosynthesis of IL‐1β by IMG cells. IMG cells secrete the mature form of IL‐1β in a caspase 1‐dependent manner. Incubation with iron provoked a greater pro‐inflammatory response. Inhibition of the iron transporter divalent metal transporter 1 protected IMG cells against Aβ‐induced inflammation. Potentiation of Aβ‐elicited IL‐1β induction by iron was also antagonized by ROS inhibitors, supporting the model that divalent metal transporter 1‐mediated iron loading and subsequent increase in ROS contribute to the inflammatory effects of Aβ in microglia. Immunoblotting and immunofluorescence microscopy indicate that iron enhances Aβ activation of NF‐κB signaling to promote IL‐1β synthesis. These results support the hypothesis that Aβ stimulates IL‐1β expression by activating NF‐κB signaling in microglia cells. Most importantly, iron appears to exacerbate the pro‐inflammatory effects of Aβ to increase IL‐1β levels. image

Over the last few years extensive studies have linked the activity of mTORC1 to lysosomal function. These observations propose an intriguing integration of cellular catabolism, sustained by lysosomes, with anabolic processes, largely controlled by mTORC1. Interestingly, lysosomal function directly affects mTORC1 activity and is regulated by ZKSCAN3 and TFEB, two transcription factors and substrates of mTORC1. Thus, the lysosomal mTOR signaling complex represents a hub of cellular energy metabolism, and its dysregulation may lead to a number of human diseases. Here, we discuss the recent developments and highlight the open questions in this growing field.

Mitophagy and mitochondrial integrated stress response (ISR) are two primary protective mechanisms to maintain functional mitochondria. Whether these two processes are coordinately regulated remains unclear. Here we show that mitochondrial fission 1 protein (Fis1), which is required for completion of mitophagy, serves as a signaling hub linking mitophagy and ISR. In mouse hepatocytes, high fat diet (HFD) feeding induces unresolved oxidative stress, defective mitophagy and enhanced type I interferon (IFN-I) response implicated in promoting metabolic inflammation. Adenoviral-mediated acute hepatic Fis1 over-expression is sufficient to reduce oxidative damage and improve glucose homeostasis in HFD fed mice. RNA-seq analysis reveals that Fis1 triggers a retrograde mitochondria-to-nucleus communication upregulating ISR genes encoding anti-oxidant defense, redox homeostasis and proteostasis pathways. Fis1-mediated ISR also suppresses expression of IFN-I stimulated genes through Atf5, which inhibits the transactivation activity of Irf3 known to control IFN-I production. Metabolite analysis demonstrates that Fis1 activation leads to accumulation of fumarate, a TCA cycle intermediate capable of increasing Atf5 activity. Consequently, hepatic Atf5 over-expression or monomethyl fumarate (MMF) treatment improves glucose homeostasis in HFD fed mice. Collectively, these results support the potential use of small molecules targeting the Fis1-Atf5 axis, such as MMF, to treat metabolic diseases.

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