Yambire KF, Rostosky C, Watanabe T, Pacheu-Grau D, Torres-Odio S, Sanchez-Guerrero A, Senderovich O, Meyron-Holtz EG, Milosevic I, Frahm J, West AP, Raimundo N. Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo. Elife. 2019 Dec 3;8 PubMed.
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The University of Adelaide
This is a fascinating and very significant study by Yambire et al. that, I think, has important implications for understanding the molecular mechanism by which mutations in the PRESENILIN genes, and possibly APP, contribute to early onset familial Alzheimer’s disease (fAD). Using HeLa cells, mouse embryonic fibroblasts, and a mouse Gaa loss-of-function mutant (modeling the lysosomal-storage disorder Glycogen storage disease II, aka Pompe disease) the authors investigated the importance of lysosomal acidification for iron homeostasis in cells.
First, by cross-comparison of various transcriptome data sets, the authors identified a relatively small number of transcription factors affected in common by inhibition of v-ATPase (using bafilomycin), the protein complex that uses energy from ATP to pump protons into lysosomes. These include important transcription factors in control of autophagy, cholesterol homeostasis, and responses to hypoxia. Further bioinformatic analysis then identified cellular responses to hypoxia as the most important processes controlled by this highly interconnected network of transcription factors.
Cellular responses to hypoxia and cellular responses to iron deficiency are so similar that iron-deficiency responses are sometimes described as “pseudo-hypoxia.” In large part, this is because a master regulator of hypoxia responses, the transcription factor HIF1, is regulated by a mechanism dependent upon both oxygen and iron (see review by Lane et al., 2015).
Yambire et al. showed that inhibition of v-ATPase in mouse fibroblasts caused stabilization of HIF1alpha (the oxygen-sensitive component of the heterodimeric HIF1 transcription factor), indicative of pseudo-hypoxia. (Related observations were made previously by Miles et al., 2017.) V-ATPase inhibition caused an overall decrease in cellular iron levels, apparently by causing retention of iron in lysosomes.
To demonstrate further that v-ATPase inhibition was causing HIF1alpha stabilization due to iron deficiency, Yambire et al. provided the inhibited cells with an alternative source of iron, Fe-citrate, to promote transport of iron into cells directly across the plasma membrane. This restored HIF1alpha to its normally less-stable state.
The authors then explored whether other disturbances of lysosomal function could cause functional iron deficiency. They analyzed fibroblasts from Gaa knockout mice and saw very significant decreases in ferrous iron levels (Fe2+, the biologically active form) and significant increases in ferric iron levels (Fe3+, the form in which iron is initially imported into cells via the endo-lysosomal pathway). Presumably, the ferric iron is accumulating in lysosomes because expression of the cytosolic ferric iron storage protein, ferritin light chain, FTL1, was decreased.
Ferrous iron is very important for the function of mitochondria to maintain cellular energy production. Yambire et al. showed that lysosomal inhibition in fibroblasts restricted mitochondrial oxygen consumption (i.e., for ATP production via oxidative phosphorylation) and this was due both to inhibition of oxidative phosphorylation and to decreased mitochondrial biogenesis. Reactive oxygen species generation increased greatly and all these effects could be ameliorated by provision of iron via Fe-citrate.
When inhibiting v-ATPase function in fibroblasts, the authors noted decreased cell proliferation and an increase in cell death. The cell death did not appear to occur via apoptosis, necroptosis, or ferroptosis. The non-apoptotic nature of the cell death led them to investigate whether iron deficiency might trigger sterile inflammatory responses. By treating mouse primary cortical neurons with the iron chelator deferoxamine, they indeed saw upregulation of innate immune regulatory genes.
The lysosomal storage disorder Glycogen storage disease II predominantly affects muscular function. However, defective lysosomal acidification previously has been associated with neurodegenerative disorders (e.g. Lee et al.’s demonstration in 2010 that fAD mutations in PSEN1 affect v-ATPase maturation and lysosomal acidification). For this and other reasons, Yambire et al. analyzed the brains of Gaa knockout mice to see the effects on iron homeostasis, mitochondrial function, and inflammation. They saw a number of phenomena indicating functional iron deficiency (increased Tfrc mRNA expression, decreased FTL1 protein expression, decreased mitochondrial complex IV activity and, interestingly, decreased choline-containing compounds). Myelination, an iron-dependent process, was affected. Importantly, cortical mitochondrial genome numbers were lower than normal at birth and continued to decline with age. Also, genes indicating inflammatory responses were upregulated from as early as 2 months of age. Stunningly, when newly weaned mice were fed an iron-enriched diet, mitochondrial genome copy numbers increased and the inflammatory markers disappeared. The stride length of the mice (indicative of motor impairment) became normal.
The importance of Yambire et al.’s observations is that they show the tight relationship between lysosomal acidification and iron homeostasis, mitochondrial biogenesis, energy production, oxidative stress, and inflammation—all recognized as important factors in AD. These observations mesh very nicely with our proposal in 2018 that a common, unifying effect of fAD mutations in the PRESENILIN genes and APP may be iron dyshomeostasis (Lumsden et al., 2018). Also, in 2019 we published evidence supporting that a heterozygous, endogenous, fAD-like mutation in the PSEN1-orthologous gene of zebrafish affects ATP synthesis and lysosomal acidification in young adult brains, consistent with such mutations causing iron dyshomeostasis (Newman et al. 2019). Since then, we have obtained additional data from our fAD-like mutant fish supporting brain iron dyshomeostasis as an early stress in fAD (not yet published). Yambire et al.’s results suggest the possibility that childhood dementias due to lysosomal storage disorders, such as Sanfilippo syndrome, and early adulthood-onset forms of fAD may share iron dyshomeostasis as a common cellular stress driving these pathologies, and that amelioration of cellular ferrous iron deficiency could prove therapeutic.
References:
Lane DJ, Merlot AM, Huang ML, Bae DH, Jansson PJ, Sahni S, Kalinowski DS, Richardson DR. Cellular iron uptake, trafficking and metabolism: Key molecules and mechanisms and their roles in disease. Biochim Biophys Acta. 2015 May;1853(5):1130-44. Epub 2015 Feb 4 PubMed.
Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, Wolfe DM, Martinez-Vicente M, Massey AC, Sovak G, Uchiyama Y, Westaway D, Cuervo AM, Nixon RA. Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations. Cell. 2010 Jun 25;141(7):1146-58. PubMed.
Lumsden AL, Rogers JT, Majd S, Newman M, Sutherland GT, Verdile G, Lardelli M. Dysregulation of Neuronal Iron Homeostasis as an Alternative Unifying Effect of Mutations Causing Familial Alzheimer's Disease. Front Neurosci. 2018;12:533. Epub 2018 Aug 13 PubMed.
Miles AL, Burr SP, Grice GL, Nathan JA. The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels. Elife. 2017 Mar 15;6 PubMed.
Newman M, Hin N, Pederson S, Lardelli M. Brain transcriptome analysis of a familial Alzheimer's disease-like mutation in the zebrafish presenilin 1 gene implies effects on energy production. Mol Brain. 2019 May 3;12(1):43. PubMed.
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