Leng L, Yuan Z, Pan R, Su X, Wang H, Xue J, Zhuang K, Gao J, Chen Z, Lin H, Xie W, Li H, Chen Z, Ren K, Zhang X, Wang W, Jin ZB, Wu S, Wang X, Yuan Z, Xu H, Chow HM, Zhang J. Microglial hexokinase 2 deficiency increases ATP generation through lipid metabolism leading to β-amyloid clearance. Nat Metab. 2022 Oct;4(10):1287-1305. Epub 2022 Oct 6 PubMed. Correction.

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  1. This is a very interesting, as well as a very complex, paper. It is worth keeping in mind that its attempts to define mechanisms derive from models, and the fidelity between the models and common AD is unclear. Partly for this reason, it is hard to know what, in the way of microglial changes, is truly adaptive versus disease-driving. That point aside, the study is commendable on several fronts, including its spatial focus on brain microglia.

    A number of the reported findings make sense, such as the switch-over to lipid oxidation when confronted by a block in the proximal glycolysis pathway. It also emphasizes what I suspect are a number of fundamental points, the key one being that the pathways that make or break energy are all interconnected. If you alter one, others will change, and glucose, amino acid, and fatty-acid-fueled bioenergetics are intertwined. Another fundamental point, noted by others, is that microglia are known to have different functional states that are determined by their bioenergetic infrastructures. The changes reported in this study tend to fit in with what is known about microglial mitochondrial phenotypes, such as the M0, M1, and M2 mitochondrial phenotypes. In that regard, I would have liked to have seen data that informed the state of mitochondria in the microglia assessed, as well as more data on the state of the pentose phosphate shunt.

    In sum, this is a really nice study that answers some questions while raising others. I certainly hope it will help to further focus the field on the relevance of energy metabolism in AD, whether it is in neurons, astrocytes, or microglia.

    View all comments by Russell Swerdlow

  2. This study by Leng and colleagues uses in vitro and in vivo models of Alzheimer’s disease to examine the impact of Aβ on microglial metabolic activity, which is an underexamined area of neurodegenerative research. Interestingly, they found that Aβ pathology increases microglial Hexokinase 2 (HK2, a key enzyme in glycolysis), which impacts microglial migration and phagocytosis. Indeed, genetic depletion or pharmacological inhibition of HK2 resulted in a significant reduction in Aβ deposition throughout the brains of their 5X-FAD mouse model. Strikingly, just seven days of daily treatment with HK2 inhibitors also reduced Aβ levels throughout the brain. I found these results to be particularly interesting, because while many groups have shown the importance of metabolic intermediates on macrophage function (Mills et al., 2016; Liu et al., 2017), how microglia adapt their metabolic status in neurodegenerative diseases and the impact it has on cellular function is still relatively unknown.

    Leng and colleagues have addressed this experimental question really well and I commend their efforts to confirm the role of HK2 in AD. They set up an extensive dataset comparing in vitro with in vivo findings, even showing how HK2 changes with age and the severity of AD pathology, in addition to comparing murine and human disease models. They also ensured that genetic depletion of HK2 matched the pharmacological inhibition of this enzyme, using not just one but two different inhibitors of HK2. While real-time metabolic assays using an instrument such as the Seahorse Bioanalyzer also would have added value to their findings, Leng and colleagues used a combination of metabolomics and glucose isotope flux analysis to confirm that HK2 regulates the switch between glycolysis and fatty acid metabolism.

    HK-dependent glycolysis can mediate NLRP3 inflammasome activation and, correspondingly, NLRP3 activation can increase the levels of HK and other glycolysis enzymes (Moon et al., 2016; Finucane et al., 2019). Since Aβ is a well-established trigger of the microglial NLRP3 inflammasome (Halle et al., 2008; Heneka et al., 2013; McManus, 2022), it is surprising that the authors did not determine whether NLRP3, or its components, such as ASC or cleaved-caspase-1, were altered in their models. Leng et al. did observe a reduction in the protein levels of IL-1β, thus, whether this was due to altered cell priming or reduced inflammasome activity would be important to establish.

    Because HK2 depletion or inhibition was so effective at reducing Aβ pathology, this would have extensive downstream consequences, reducing long-term microglial activation by removing the very trigger (i.e., Aβ) that chronically activates the cells. Future work could examine this in detail to determine if daily inhibition of HK2 is necessary to maintain these effects, and whether targeting HK2 in advanced disease stages is still effective at preventing or rescuing cognitive decline. As one of the HK inhibitors used in this study is already in clinical trials (Lonidamine, as a cancer therapy), the results of extended datasets at later disease stages would be very exciting for scientists and patients alike.

    References:

    Mills EL, Kelly B, Logan A, Costa AS, Varma M, Bryant CE, Tourlomousis P, Däbritz JH, Gottlieb E, Latorre I, Corr SC, McManus G, Ryan D, Jacobs HT, Szibor M, Xavier RJ, Braun T, Frezza C, Murphy MP, O'Neill LA. Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages. Cell. 2016 Oct 6;167(2):457-470.e13. Epub 2016 Sep 22 PubMed.

    Liu PS, Wang H, Li X, Chao T, Teav T, Christen S, Di Conza G, Cheng WC, Chou CH, Vavakova M, Muret C, Debackere K, Mazzone M, Huang HD, Fendt SM, Ivanisevic J, Ho PC. α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat Immunol. 2017 Sep;18(9):985-994. Epub 2017 Jul 17 PubMed.

    Moon JS, Hisata S, Park MA, DeNicola GM, Ryter SW, Nakahira K, Choi AM. mTORC1-Induced HK1-Dependent Glycolysis Regulates NLRP3 Inflammasome Activation. Cell Rep. 2015 Jul 7;12(1):102-115. Epub 2015 Jun 25 PubMed.

    Finucane OM, Sugrue J, Rubio-Araiz A, Guillot-Sestier MV, Lynch MA. The NLRP3 inflammasome modulates glycolysis by increasing PFKFB3 in an IL-1β-dependent manner in macrophages. Sci Rep. 2019 Mar 11;9(1):4034. PubMed.

    Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, Fitzgerald KA, Latz E, Moore KJ, Golenbock DT. The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol. 2008 Aug;9(8):857-65. PubMed.

    Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, Griep A, Axt D, Remus A, Tzeng TC, Gelpi E, Halle A, Korte M, Latz E, Golenbock DT. NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice. Nature. 2013 Jan 31;493(7434):674-8. Epub 2012 Dec 19 PubMed.

    McManus RM. The Role of Immunity in Alzheimer's Disease. Adv Biol (Weinh). 2022 May;6(5):e2101166. Epub 2022 Mar 7 PubMed.

    View all comments by Róisín McManus

  3. This new report by Leng et al. is an exciting new addition to a growing number of studies implicating the regulation of microglia metabolism as a central driver in Alzheimer’s disease pathogenesis.

    The authors first show cell-specific expression of the various hexokinase (HK) isoforms within the mouse and human AD brain, with microglia predominantly expressing the HK2 isoform. They then conditionally knock out HK2 in microglia (and macrophages) using CX3CR1-Cre. Remarkably, this reduced plaque numbers by ~half when crossed to the 5XFAD model of amyloidosis. Importantly, this elimination of HK2 in microglia led to an increase in synaptic markers and a complete rescue of 5XFAD-associated cognitive deficits.

    Excitingly, treatment with HK inhibitors, and in particular the HK2-specific inhibitor 3-bromopyruvate (3-BP), showed similar protective effects. This prompted the authors to move in vitro, where they show that HK2 inhibition results in an increase in microglial phagocytosis (an energetically demanding process). Because hexokinase catalyzes the first step of glucose metabolism, inhibition of the enzyme should reduce ATP levels in the cell (as shown previously in multiple cell types). Paradoxically, Leng et al. show the opposite effect in primary microglia and BV2 cells. The authors go on to suggest that this increase in ATP is due to transcriptional activation of lipid metabolism and increased fatty acid beta-oxidation, perhaps downstream of an upregulation of the DAM gene lipoprotein lipase (Lpl). This is also quite interesting given that the basic tenets of immunometabolism state that “homeostatic” myeloid cells rely heavily on fatty acid beta-oxidation.

    This study comes as the field gains a new appreciation for the metabolic demands of microglia (Xiang et al., 2021), and adds to a growing body of literature implicating the microglial metabolic response to plaques as a contributing event in AD pathogenesis (Baik et al., 2019; March-Diaz et al., 2021; Grubman et al., 2021; Nguyen et al., 2020). This includes studies implicating AD risk genes such as TREM2 and APO4E, further highlighting the importance of microglial metabolism in the onset and progression of AD (Konttinen et al., 2019; Lee et al., 2022). 

    References:

    Xiang X, Wind K, Wiedemann T, Blume T, Shi Y, Briel N, Beyer L, Biechele G, Eckenweber F, Zatcepin A, Lammich S, Ribicic S, Tahirovic S, Willem M, Deussing M, Palleis C, Rauchmann BS, Gildehaus FJ, Lindner S, Spitz C, Franzmeier N, Baumann K, Rominger A, Bartenstein P, Ziegler S, Drzezga A, Respondek G, Buerger K, Perneczky R, Levin J, Höglinger GU, Herms J, Haass C, Brendel M. Microglial activation states drive glucose uptake and FDG-PET alterations in neurodegenerative diseases. Sci Transl Med. 2021 Oct 13;13(615):eabe5640. PubMed.

    Baik SH, Kang S, Lee W, Choi H, Chung S, Kim JI, Mook-Jung I. A Breakdown in Metabolic Reprogramming Causes Microglia Dysfunction in Alzheimer's Disease. Cell Metab. 2019 Sep 3;30(3):493-507.e6. Epub 2019 Jun 27 PubMed.

    March-Diaz R, Lara-Ureña N, Romero-Molina C, Heras-Garvin A, Ortega-de San Luis C, Alvarez-Vergara MI, Sanchez-Garcia MA, Sanchez-Mejias E, Davila JC, Rosales-Nieves AE, Forja C, Navarro V, Gomez-Arboledas A, Sanchez-Mico MV, Viehweger A, Gerpe A, Hodson EJ, Vizuete M, Bishop T, Serrano-Pozo A, Lopez-Barneo J, Berra E, Gutierrez A, Vitorica J, Pascual A. Hypoxia compromises the mitochondrial metabolism of Alzheimer’s disease microglia via HIF1. Nature Aging. 1, 2021, pp.385–99. Nat. Aging.

    Grubman A, Choo XY, Chew G, Ouyang JF, Sun G, Croft NP, Rossello FJ, Simmons R, Buckberry S, Landin DV, Pflueger J, Vandekolk TH, Abay Z, Zhou Y, Liu X, Chen J, Larcombe M, Haynes JM, McLean C, Williams S, Chai SY, Wilson T, Lister R, Pouton CW, Purcell AW, Rackham OJ, Petretto E, Polo JM. Transcriptional signature in microglia associated with Aβ plaque phagocytosis. Nat Commun. 2021 May 21;12(1):3015. PubMed.

    Nguyen AT, Wang K, Hu G, Wang X, Miao Z, Azevedo JA, Suh E, Van Deerlin VM, Choi D, Roeder K, Li M, Lee EB. APOE and TREM2 regulate amyloid-responsive microglia in Alzheimer's disease. Acta Neuropathol. 2020 Oct;140(4):477-493. Epub 2020 Aug 25 PubMed. Correction.

    Konttinen H, Cabral-da-Silva ME, Ohtonen S, Wojciechowski S, Shakirzyanova A, Caligola S, Giugno R, Ishchenko Y, Hernández D, Fazaludeen MF, Eamen S, Budia MG, Fagerlund I, Scoyni F, Korhonen P, Huber N, Haapasalo A, Hewitt AW, Vickers J, Smith GC, Oksanen M, Graff C, Kanninen KM, Lehtonen S, Propson N, Schwartz MP, Pébay A, Koistinaho J, Ooi L, Malm T. PSEN1ΔE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia. Stem Cell Reports. 2019 Oct 8;13(4):669-683. Epub 2019 Sep 12 PubMed.

    Lee S, Devanney ND, Golden LR, Smith CT, Schwarz JL, Walsh AE, Clarke HA, Goulding DS, Allenger EJ, Morillo-Segovia G, Friday CM, Gorman AA, Hawkinson TR, MacLean SM, Williams HC, Sun RC, Morganti JM, Johnson LA. APOE modulates microglial immunometabolism in response to age, amyloid pathology, and inflammatory challenge. bioRxiv. May 20, 2022 bioRxiv

    View all comments by Lance Johnson

  4. The manner in which specific energy substrates impact other aspects of cell biology is complicated and fascinating. It is now quite clear that many energy substrates—including glucose—boost mTOR, the activity of which suppresses autophagy and related processes. It is important to consider these "related processes," because many of the proteins required for autophagy also play a role in LC3-associated phagocytosis and LC3-associated endocytosis, of which the latter has been shown to participate in microglial degradation of Aβ (Heckmann et al., 2019). 

    One aspect of glycolysis that has gained interest in cancer biology is its contribution to the synthesis of amino acids. Glycine and serine levels are substantially depleted when glycolysis is restricted, and such amino acids are necessary for full mTORC1 activity (Takahara et al., 2020). Thus, glycolysis inhibition may suppress mTOR and thus activate autophagy or related processes to speed Aβ removal.

    Relevant to this hypothesis is the interaction of the APOE allele with autophagy-related processes (Simonovitch et al., 2016). Based on the ability of ApoE4 to suppress an enhancer element present in many autophagy-related genes (Parcon et al., 2018; Lima et al., 2020), it might be predicted that an oxidative shift in the glycolysis-OXPHOS balance would be more effective in individuals lacking an APOE ε4 allele. And, indeed, that has been borne out in human trials Reger et al., 2004; Henderson et al., 2009; Torosyan et al., 2018). 

    References:

    Heckmann BL, Teubner BJ, Tummers B, Boada-Romero E, Harris L, Yang M, Guy CS, Zakharenko SS, Green DR. LC3-Associated Endocytosis Facilitates β-Amyloid Clearance and Mitigates Neurodegeneration in Murine Alzheimer's Disease. Cell. 2019 Jul 25;178(3):536-551.e14. Epub 2019 Jun 27 PubMed. Correction.

    Takahara T, Amemiya Y, Sugiyama R, Maki M, Shibata H. Amino acid-dependent control of mTORC1 signaling: a variety of regulatory modes. J Biomed Sci. 2020 Aug 17;27(1):87. PubMed.

    Simonovitch S, Schmukler E, Bespalko A, Iram T, Frenkel D, Holtzman DM, Masliah E, Michaelson DM, Pinkas-Kramarski R. Impaired Autophagy in APOE4 Astrocytes. J Alzheimers Dis. 2016;51(3):915-27. PubMed.

    Parcon PA, Balasubramaniam M, Ayyadevara S, Jones RA, Liu L, Shmookler Reis RJ, Barger SW, Mrak RE, Griffin WS. Apolipoprotein E4 inhibits autophagy gene products through direct, specific binding to CLEAR motifs. Alzheimers Dement. 2018 Feb;14(2):230-242. Epub 2017 Sep 22 PubMed.

    Lima D, Hacke AC, Inaba J, Pessôa CA, Kerman K. Electrochemical detection of specific interactions between apolipoprotein E isoforms and DNA sequences related to Alzheimer's disease. Bioelectrochemistry. 2020 Jun;133:107447. Epub 2019 Dec 23 PubMed.

    Reger MA, Henderson ST, Hale C, Cholerton B, Baker LD, Watson GS, Hyde K, Chapman D, Craft S. Effects of beta-hydroxybutyrate on cognition in memory-impaired adults. Neurobiol Aging. 2004 Mar;25(3):311-4. PubMed.

    Henderson ST, Vogel JL, Barr LJ, Garvin F, Jones JJ, Costantini LC. Study of the ketogenic agent AC-1202 in mild to moderate Alzheimer's disease: a randomized, double-blind, placebo-controlled, multicenter trial. Nutr Metab (Lond). 2009;6:31. PubMed.

    Torosyan N, Sethanandha C, Grill JD, Dilley ML, Lee J, Cummings JL, Ossinalde C, Silverman DH. Changes in regional cerebral blood flow associated with a 45 day course of the ketogenic agent, caprylidene, in patients with mild to moderate Alzheimer's disease: Results of a randomized, double-blinded, pilot study. Exp Gerontol. 2018 Oct 1;111:118-121. Epub 2018 Jul 10 PubMed.

    View all comments by Steve Barger

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