In neurodegenerative diseases, debris of dead neurons are thought to trigger glia-mediated neuroinflammation, thus increasing neuronal death. Here we show that the expression of neurotoxic proteins associated with these diseases in microglia alone is sufficient to directly trigger death of naive neurons and to propagate neuronal death through activation of naive astrocytes to the A1 state. Injury propagation is mediated, in great part, by the release of fragmented and dysfunctional microglial mitochondria into the neuronal milieu. The amount of damaged mitochondria released from microglia relative to functional mitochondria and the consequent neuronal injury are determined by Fis1-mediated mitochondrial fragmentation within the glial cells. The propagation of the inflammatory response and neuronal cell death by extracellular dysfunctional mitochondria suggests a potential new intervention for neurodegeneration—one that inhibits mitochondrial fragmentation in microglia, thus inhibiting the release of dysfunctional mitochondria into the extracellular milieu of the brain, without affecting the release of healthy neuroprotective mitochondria.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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In memory of Ben Barres, whose advice and research inspired this study. This work was supported, in part, by a Stanford Discovery Innovation Award and R01 HL52141 to D.M.-R., a Paul & Daisy Soros Fellowship to P.S.M., a postdoctoral fellowship from the Australian National Health and Medical Research Council (GNT1052961) and the Glenn Foundation Glenn Award to S.A.L., RO1AG058047 to K.I.A., and R35 HL135736 to G.W.D. The authors thank N. Raju and S. Avolicino for assistance with immunohistochemistry and J. Perrino for technical support with electron microscopy.
Patents on P110 and its utility in HD, ALS and other neurodegenerative diseases have been filed by D.M.‐R. and A.U.J. The other authors declare no competing interests.
Peer review information Nature Neuroscience thanks Robert Friedlander, Krista Spiller and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Integrated supplementary information
Supplementary Figure 1 Inhibition of excessive mitochondrial fission by treatment with P110 in vivo reduces sustained microglial-astrocytic activation and subsequent pro-inflammatory response in three mouse models of neurodegenerative diseases, AD, HD and ALS.
(a) Area occupied by GFAP immunoreactive astrocytes (brown) was quantified in hippocampus, striatum and spinal cord, in 5xFAD AD mice, in R6/2 HD mice and SOD1 G93A ALS mice, respectively, vehicle- or P110-treated with 3 mg/kg/day for the time indicated in (1a). (b) Area occupied by Iba-1 immunoreactive microglia (brown) was quantified in hippocampus, striatum and spinal cord, in 5xFAD AD mice, in R6/2 HD mice and SOD1 G93A ALS mice, respectively, vehicle- or P110-treated with 3 mg/kg/day for the time indicated in (1a; red rectangles). (n = 5 per group/ 2 sections per mice.). (c) Representative western blot staining for glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, Iba-1, a marker of microglial activation, NLRP3, a marker of inflammasome activation and COX-2, a marker of canonical inflammation in whole brain R6/2 HD mice at endpoint. (d) Representative western blot staining for glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, Iba-1, a marker of microglial activation, NLRP3, a marker of inflammasome activation and COX-2, a marker of canonical inflammation in spinal cord SOD1 G93A mice at endpoint. (e) Representative western blot staining for glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, Iba-1, a marker of microglial activation, NLRP3, a marker of inflammasome activation and COX-2, a marker of canonical inflammation in whole brain lysate in 5XFAD mice at endpoint. 1a. (n = 5 per group). β-actin was used as a loading control. (f) NLRP3 and (g) COX-2 protein levels (determined by quantitating immunostaining) in each mouse model, presented as ratio to the corresponding WT mice. Data were evaluated by one-way ANOVA and Tukey’s multiple comparisons test for multiple testing between each treatment group. All graphs represent mean ± s.d.
Supplementary Figure 2 Excessive mitochondrial fission and dysfunction linked with inflammasome activation in microglia.
(a) Schematic of the experimental design using primary rat microglia treated with LPS (10 ng/ml) for 3 hours, followed by nigericin (1.2µM)-treatment in the presence/ absence of P110 (1 µM added 15 minutes prior to LPS) for 21 hours in serum and antibiotic free DMEM (top). Cellular levels of phospho-p44/42, phosphorylated NFκB and phosphorylated c-Jun N-terminal kinase (JNK) were determined by immunoblotting, quantified and presented as fold-change of control in rat primary microglia; (n=3). (b) Levels of Il-1β and caspase1-p20 were determined in cultured media of these primary microglia by immunoblotting. β-actin in the cells from the corresponding cultures was used as a cell loading control; (n = 2.) (c) Mitochondrial NLRP3 and ASC levels were determined by immunoblotting of mitochondrial fraction, quantified and presented as fold-change of control in these primary microglia. Data were evaluated by one-way ANOVA and Holm-Sidak’s multiple comparisons test for multiple testing between each treatment group; (n=5) All graphs represent mean ± s.d.
Supplementary Figure 3 Transfer of conditioned media of primary rat microglia activates primary rat astrocytes in vitro in a process dependent on Drp1/Fis1 interaction in the microglia and results in Drp1 activation and mitochondrial dysfunction in the astrocytes.
(a) The protocol of cell treatments and media transfer (above). Levels of GFAP protein were determined in cell extracts by immunoblotting rat primary astrocytes treated with supernatant from activated rat primary microglia (MCM); β-actin was used as a loading control; n = 4 (b) IL-1α, IL-1β, TNFα in the culture media of the astrocytes were measured by ELISA; n = 6 and (c) C1q RNA levels in cell extracts were measured by qPCR in cells treated as described in a; n = 5 (d) Drp1 phosphorylation in astrocytes (as a measure of Drp1 activation) were determined by immunoblotting in primary astrocytes treated with supernatant from activated primary microglia; β-actin was used as a loading control; n=4. (e) Mitochondrial levels of NLRP3 and ASC were determined by immunoblotting in the primary astrocytes; β-actin was used as a loading control. Protein levels were quantified and are presented as fold-change of control; n = 3-6. (f) Quantitative PCR analysis of astrocyte-specific phagocytic receptors (multiple EGF-like domains 10, Megf10, and MER proto-oncogene, tyrosine kinase, Mertk) and bridging molecules (receptor tyrosine kinase, Axl, and its ligand, growth arrest-specific 6, Gas6) in primary astrocytes treated with conditioned media, as in a; n=4. (g) OXPHOS in primary astrocytes treated with conditioned media, as in a; n=4. Data were evaluated by one-way ANOVA and Holm-Sidak’s multiple comparisons test for multiple testing between each treatment group. All graphs represent mean ± s.d.
(a) Primary cortical rat neurons were treated with conditioned media of primary rat microglia treated as in described in Fig. 2l for 24 hours without (Con) or with LPS/Nig without (Veh) or with P110 (1 μM) and assessed for survival, using LDH release from the neurons. (n=12) (b) Protocol of injury propagation from primary rat microglia activated by LPS to primary cortical rat neurons by media from activated astrocytes (aACM), as in described in Fig. 7a. Where indicated, mitochondria were from the astrocytic media (aACM; filtration through 0.2-μm filters; MitoΔ) and neuronal cell death was determined using LDH release from the neurons. Removal of mitochondria from aACM decreased neuronal survival, indicating the neuroprotective role of functional extracellular mitochondria; (n=10). (c) Primary cortical rat neurons were treated with conditioned media of primary rat astrocytes treated as in described in Fig. 6j for 24 hours. Neuronal cell death was determined using LDH release from the neurons; n=5). (d) Neuroinflammation response propagated from activated microglia to astrocytes to cause cell death of naïve neurons in models of neurodegenerative diseases via expelled and damaged glial mitochondria. Both functional and dysfunctional mitochondria are expelled from both astrocytes and microglia cells. However, mitochondrial dysfunction in microglia triggers the release of more dysfunctional mitochondria relative to functional mitochondria, and these dysfunctional extracellular mitochondria propagate the injurious signal to neurons directly, as well as through inducing mitochondrial dysfunction, activation and neuroinflammation in the astrocytes (left). Inhibiting Drp1/Fis1-mediated pathological fission in the microglia, using a selective inhibitor, such as P110, greatly reduces neuroinflammation, propagation of mitochondrial dysfunction and neuronal cell death. Blocking Drp1Fis1-mediated mitochondrial fission in the microglia increased the transfer benefit to subsequent cell types, in part, by releasing healthier mitochondria into the neuronal milieu (right). Whether the neurons uptake the extracellular mitochondria remains to be determined. Data were evaluated by one-way ANOVA and Holm-Sidak’s multiple comparisons test for multiple testing between each treatment group. All graphs represent mean ± s.d.
Images acquired were processed using a macro in Fiji (Image J).
Source Data for Western Blots.
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Science Translational Medicine (2019)