Activity-dependent tau cleavage by caspase-3 promotes neuronal dysfunction and synaptotoxicity.

Tau-mediated toxicity is associated with cognitive decline and Alzheimer's disease (AD) progression. In particular, tau post-translational modifications (PTMs) are thought to generate aberrant tau species resulting in neuronal dysfunction. Despite being well characterized in postmortem AD brain, it is unclear how caspase-mediated C-terminal tau cleavage promotes neurodegeneration, as few studies have developed the models to dissect this pathogenic mechanism. Here, we show that proteasome impairment results in cleaved tau accumulation at the post-synaptic density (PSD), a process that is modulated by neuronal activity. Cleaved tau (at residue D421) impairs neuronal firing and causes inefficient initiation of network bursts, consistent with reduced excitatory drive. We propose that reduced neuronal activity, or silencing, is coupled to proteasome dysfunction, which drives cleaved tau accumulation at the PSD and subsequent synaptotoxicity. Our study connects three common themes in the progression of AD: impaired proteostasis, caspase-mediated tau cleavage, and synapse degeneration.

Manganese-induced Mitochondrial Dysfunction Is Not Detectable at Exposures Below the Acute Cytotoxic Threshold in Neuronal Cell Types.

Manganese (Mn) is an essential metal, but excessive exposures have been well-documented to culminate in neurotoxicity. Curiously, the precise mechanisms of Mn neurotoxicity are still unknown. One hypothesis suggests that Mn exerts its toxicity by inhibiting mitochondrial function, which then (if exposure levels are high and long enough) leads to cell death. Here, we used a Huntington's disease cell model with known differential sensitivities to manganese-STHdhQ7/Q7 and STHdhQ111/Q111 cells-to examine the effects of acute Mn exposure on mitochondrial function. We determined toxicity thresholds for each cell line using both changes in cell number and caspase-3/7 activation. We used a range of acute Mn exposures (0-300 µM), both above and below the cytotoxic threshold, to evaluate mitochondria-associated metabolic balance, mitochondrial respiration, and substrate dependence. In both cell lines, we observed no effect on markers of mitochondrial function at subtoxic Mn exposures (below detectable levels of cell death), yet at supratoxic exposures (above detectable levels of cell death) mitochondrial function significantly declined. We validated these findings in primary striatal neurons. In cell lines, we further observed that subtoxic Mn concentrations do not affect glycolytic function or major intracellular metabolite quantities. These data suggest that in this system, Mn exposure impairs mitochondrial function only at concentrations coincident with or above the initiation of cell death and is not consistent with the hypothesis that mitochondrial dysfunction precedes or induces Mn cytotoxicity.

Manganese Acts upon Insulin/IGF Receptors to Phosphorylate AKT and Increase Glucose Uptake in Huntington's Disease Cells.

Perturbations in insulin/IGF signaling and manganese (Mn2+) uptake and signaling have been separately reported in Huntington's disease (HD) models. Insulin/IGF supplementation ameliorates HD phenotypes via upregulation of AKT, a known Mn2+-responsive kinase. Limited evidence both in vivo and in purified biochemical systems suggest Mn2+ enhances insulin/IGF receptor (IR/IGFR), an upstream tyrosine kinase of AKT. Conversely, Mn2+ deficiency impairs insulin release and associated glucose tolerance in vivo. Here, we test the hypothesis that Mn2+-dependent AKT signaling is predominantly mediated by direct Mn2+ activation of the insulin/IGF receptors, and HD-related impairments in insulin/IGF signaling are due to HD genotype-associated deficits in Mn2+ bioavailability. We examined the combined effects of IGF-1 and/or Mn2+ treatments on AKT signaling in multiple HD cellular models. Mn2+ treatment potentiates p-IGFR/IR-dependent AKT phosphorylation under physiological (1 nM) or saturating (10 nM) concentrations of IGF-1 directly at the level of intracellular activation of IGFR/IR. Using a multi-pharmacological approach, we find that > 70-80% of Mn2+-associated AKT signaling across rodent and human neuronal cell models is specifically dependent on IR/IGFR, versus other signaling pathways upstream of AKT activation. Mn2+-induced p-IGFR and p-AKT were diminished in HD cell models, and, consistent with our hypothesis, were rescued by co-treatment of Mn2+ and IGF-1. Lastly, Mn2+-induced IGF signaling can modulate HD-relevant biological processes, as the reduced glucose uptake in HD STHdh cells was partially reversed by Mn2+ supplementation. Our data demonstrate that Mn2+ supplementation increases peak IGFR/IR-induced p-AKT likely via direct effects on IGFR/IR, consistent with its role as a cofactor, and suggests reduced Mn2+ bioavailability contributes to impaired IGF signaling and glucose uptake in HD models.

Dysregulation of TFEB contributes to manganese-induced autophagic failure and mitochondrial dysfunction in astrocytes.

Epidemiological and clinical studies have long shown that exposure to high levels of heavy metals are associated with increased risks of neurodegenerative diseases. It is widely accepted that autophagic dysfunction is involved in pathogenesis of various neurodegenerative disorders; however, the role of heavy metals in regulation of macroautophagy/autophagy is unclear. Here, we show that manganese (Mn) induces a decline in nuclear localization of TFEB (transcription factor EB), a master regulator of the autophagy-lysosome pathway, leading to autophagic dysfunction in astrocytes of mouse striatum. We further show that Mn exposure suppresses autophagic-lysosomal degradation of mitochondria and induces accumulation of unhealthy mitochondria. Activation of autophagy by rapamycin or TFEB overexpression ameliorates Mn-induced mitochondrial respiratory dysfunction and reactive oxygen species (ROS) generation in astrocytes, suggesting a causal relation between autophagic failure and mitochondrial dysfunction in Mn toxicity. Taken together, our data demonstrate that Mn inhibits TFEB activity, leading to impaired autophagy that is causally related to mitochondrial dysfunction in astrocytes. These findings reveal a previously unappreciated role for Mn in dysregulation of autophagy and identify TFEB as a potential therapeutic target to mitigate Mn toxicity. ABBREVIATIONS: BECN1: beclin 1; CTSD: cathepsin D; DMEM: Dulbecco's Modified Eagle Medium; GFAP: glial fibrillary acid protein; GFP: green fluorescent protein; HBSS: hanks balanced salt solution; LAMP: lysosomal-associated membrane protein; LDH: lactate dehydrogenase; Lys Inh: lysosomal inhibitors; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; Mn: manganese; MTOR: mechanistic target of rapamycin kinase; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PFA: paraformaldehyde; PI: propidium iodide; ROS: reactive oxygen species; s.c.: subcutaneous; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB.

Acute manganese treatment restores defective autophagic cargo loading in Huntington's disease cell lines.

The molecular etiology linking the pathogenic mutations in the Huntingtin (Htt) gene with Huntington's disease (HD) is unknown. Prior work suggests a role for Htt in neuronal autophagic function and mutant HTT protein disrupts autophagic cargo loading. Reductions in the bioavailability of the essential metal manganese (Mn) are seen in models of HD. Excess cellular Mn impacts autophagic function, but the target and molecular basis of these changes are unknown. Thus, we sought to determine if changes in cellular Mn status impact autophagic processes in a wild-type or mutant Htt-dependent manner. We report that the HD genotype is associated with reduced Mn-induced autophagy and that acute Mn exposure increases autophagosome induction/formation. To determine if a deficit in bioavailable Mn is mechanistically linked to the autophagy-related HD cellular phenotypes, we examined autophagosomes by electron microscopy. We observed that a 24 h 100 uM Mn restoration treatment protocol attenuated an established HD 'cargo-recognition failure' in the STHdh HD model cells by increasing the percentage of filled autophagosomes. Mn restoration had no effect on HTT aggregate number, but a 72 h co-treatment with chloroquine (CQ) in GFP-72Q-expressing HEK293 cells increased the number of visible aggregates in a dose-dependent manner. As CQ prevents autophagic degradation this indicates that Mn restoration in HD cell models facilitates incorporation of aggregates into autophagosomes. Together, these findings suggest that defective Mn homeostasis in HD models is upstream of the impaired autophagic flux and provide proof-of-principle support for increasing bioavailable Mn in HD to restore autophagic function and promote aggregate clearance.

Phosphatidylinositol 3 kinase (PI3K) modulates manganese homeostasis and manganese-induced cell signaling in a murine striatal cell line.

In a recent study, we found that blocking the protein kinase ataxia telangiectasia mutated (ATM) with the small molecule inhibitor (SMI) KU-55933 can completely abrogate Mn-induced phosphorylation of p53 at serine 15 (p-p53) in human induced pluripotent stem cell (hiPSC)-differentiated striatal neuroprogenitors. However, in the immortalized mouse striatal progenitor cell line STHdhQ7/Q7, a concentration of KU55933 far exceeding its IC50 for ATM was required to inhibit Mn-induced p-p53. This suggested an alternative signaling system redundant with ATM kinase for activating p53 in this cell line- one that was altered by KU55933 at these higher concentrations (i.e. mTORC1, DNApk, PI3K). To test the hypothesis that one or more of these signaling pathways contributed to Mn-induced p-p53, we utilized a set of SMIs (e.g. NU7441 and LY294002) known to block DNApk, PI3K, and mTORC1 at distinct concentrations. We found that the SMIs inhibit Mn-induced p-p53 expression near the expected IC50s for PI3K, versus other known targets. We hypothesized that inhibiting PI3K reduces intracellular Mn and thereby decreases activation of p53 by Mn. Using the cellular fura-2 manganese extraction assay (CFMEA), we determined that KU55933/60019, NU7441, and LY294002 (at concentrations near their IC50s for PI3K) all decrease intracellular Mn (∼50%) after a dual, 24-h Mn and SMI exposure. Many pathways are activated by Mn aside from p-p53, including AKT and mTOR pathways. Thus, we explored the activation of these pathways by Mn in STHdh cells as well as the effects of other pathway inhibitors. p-AKT and p-S6 activation by Mn is almost completely blocked upon addition of NU7441(5µM) or LY294002(7µM), supporting PI3K's upstream role in the AKT/mTOR pathway. We also investigated whether PI3K inhibition blocks Mn uptake in other cell lines. LY294002 exposure did not reduce Mn uptake in ST14A, Neuro2A, HEK293, MEF, or hiPSC-derived neuroprogenitors. Next, we sought to determine whether inhibition of PI3K blocked p53 phosphorylation by directly blocking an unknown PI3K/p53 interaction or indirectly reducing intracellular Mn, decreasing p-p53 expression. In-Cell Western and CFMEA experiments using multiple concentrations of Mn exposures demonstrated that intracellular Mn levels directly correlated with p-p53 expression with or without addition of LY294002. Finally, we examined whether PI3K inhibition was able to block Mn-induced p-p53 activity in hiPSC-derived striatal neuroprogenitors. As expected, LY294002 does not block Mn-induced p-p53 as PI3K inhibition is unable to reduce Mn net uptake in this cell line, suggesting the effect of LY294002 on Mn uptake is relatively specific to the STHdh mouse striatal cell line.

Manganese and the Insulin-IGF Signaling Network in Huntington's Disease and Other Neurodegenerative Disorders.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting in motor impairment and death in patients. Recently, several studies have demonstrated insulin or insulin-like growth factor (IGF) treatment in models of HD, resulting in potent amelioration of HD phenotypes via modulation of the PI3K/AKT/mTOR pathways. Administration of IGF and insulin can rescue microtubule transport, metabolic function, and autophagy defects, resulting in clearance of Huntingtin (HTT) aggregates, restoration of mitochondrial function, amelioration of motor abnormalities, and enhanced survival. Manganese (Mn) is an essential metal to all biological systems but, in excess, can be toxic. Interestingly, several studies have revealed the insulin-mimetic effects of Mn-demonstrating Mn can activate several of the same metabolic kinases and increase peripheral and neuronal insulin and IGF-1 levels in rodent models. Separate studies have shown mouse and human striatal neuroprogenitor cell (NPC) models exhibit a deficit in cellular Mn uptake, indicative of a Mn deficiency. Furthermore, evidence from the literature reveals a striking overlap between cellular consequences of Mn deficiency (i.e., impaired function of Mn-dependent enzymes) and known HD endophenotypes including excitotoxicity, increased reactive oxygen species (ROS) accumulation, and decreased mitochondrial function. Here we review published evidence supporting a hypothesis that (1) the potent effect of IGF or insulin treatment on HD models, (2) the insulin-mimetic effects of Mn, and (3) the newly discovered Mn-dependent perturbations in HD may all be functionally related. Together, this review will present the intriguing possibility that intricate regulatory cross-talk exists between Mn biology and/or toxicology and the insulin/IGF signaling pathways which may be deeply connected to HD pathology and, perhaps, other neurodegenerative diseases (NDDs) and other neuropathological conditions.

Reduced bioavailable manganese causes striatal urea cycle pathology in Huntington's disease mouse model.

Huntington's disease (HD) is caused by a mutation in the huntingtin gene (HTT), resulting in profound striatal neurodegeneration through an unknown mechanism. Perturbations in the urea cycle have been reported in HD models and in HD patient blood and brain. In neurons, arginase is a central urea cycle enzyme, and the metal manganese (Mn) is an essential cofactor. Deficient biological responses to Mn, and reduced Mn accumulation have been observed in HD striatal mouse and cell models. Here we report in vivo and ex vivo evidence of a urea cycle metabolic phenotype in a prodromal HD mouse model. Further, either in vivo or in vitro Mn supplementation reverses the urea-cycle pathology by restoring arginase activity. We show that Arginase 2 (ARG2) is the arginase enzyme present in these mouse brain models, with ARG2 protein levels directly increased by Mn exposure. ARG2 protein is not reduced in the prodromal stage, though enzyme activity is reduced, indicating that altered Mn bioavailability as a cofactor leads to the deficient enzymatic activity. These data support a hypothesis that mutant HTT leads to a selective deficiency of neuronal Mn at an early disease stage, contributing to HD striatal urea-cycle pathophysiology through an effect on arginase activity.

Genomic Instability Associated with p53 Knockdown in the Generation of Huntington's Disease Human Induced Pluripotent Stem Cells.

Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.

Metabolic Catastrophe in Mice Lacking Transferrin Receptor in Muscle.

Transferrin receptor (Tfr1) is ubiquitously expressed, but its roles in non-hematopoietic cells are incompletely understood. We used a tissue-specific conditional knockout strategy to ask whether skeletal muscle required Tfr1 for iron uptake. We found that iron assimilation via Tfr1 was critical for skeletal muscle metabolism, and that iron deficiency in muscle led to dramatic changes, not only in muscle, but also in adipose tissue and liver. Inactivation of Tfr1 incapacitated normal energy production in muscle, leading to growth arrest and a muted attempt to switch to fatty acid ß oxidation, using up fat stores. Starvation signals stimulated gluconeogenesis in the liver, but amino acid substrates became limiting and hypoglycemia ensued. Surprisingly, the liver was also iron deficient, and production of the iron regulatory hormone hepcidin was depressed. Our observations reveal a complex interaction between iron homeostasis and metabolism that has implications for metabolic and iron disorders.

A novel manganese-dependent ATM-p53 signaling pathway is selectively impaired in patient-based neuroprogenitor and murine striatal models of Huntington's disease.

The essential micronutrient manganese is enriched in brain, especially in the basal ganglia. We sought to identify neuronal signaling pathways responsive to neurologically relevant manganese levels, as previous data suggested that alterations in striatal manganese handling occur in Huntington's disease (HD) models. We found that p53 phosphorylation at serine 15 is the most responsive cell signaling event to manganese exposure (of 18 tested) in human neuroprogenitors and a mouse striatal cell line. Manganese-dependent activation of p53 was severely diminished in HD cells. Inhibitors of ataxia telangiectasia mutated (ATM) kinase decreased manganese-dependent phosphorylation of p53. Likewise, analysis of ATM autophosphorylation and additional ATM kinase targets, H2AX and CHK2, support a role for ATM in the activation of p53 by manganese and that a defect in this process occurs in HD. Furthermore, the deficit in Mn-dependent activation of ATM kinase in HD neuroprogenitors was highly selective, as DNA damage and oxidative injury, canonical activators of ATM, did not show similar deficits. We assessed cellular manganese handling to test for correlations with the ATM-p53 pathway, and we observed reduced Mn accumulation in HD human neuroprogenitors and HD mouse striatal cells at manganese exposures associated with altered p53 activation. To determine if this phenotype contributes to the deficit in manganese-dependent ATM activation, we used pharmacological manipulation to equalize manganese levels between HD and control mouse striatal cells and rescued the ATM-p53 signaling deficit. Collectively, our data demonstrate selective alterations in manganese biology in cellular models of HD manifest in ATM-p53 signaling.