Octopamine metabolically reprograms astrocytes to confer neuroprotection against α-synuclein

Abstract

Octopamine is a well-established invertebrate neurotransmitter involved in fight or flight responses. In mammals, its function was replaced by epinephrine. Nevertheless, it is present at trace amounts and can modulate the release of monoamine neurotransmitters by a yet unidentified mechanism. Here, through a multidisciplinary approach utilizing in vitro and in vivo models of α-synucleinopathy, we uncovered an unprecedented role for octopamine in driving the conversion from toxic to neuroprotective astrocytes in the cerebral cortex by fostering aerobic glycolysis. Physiological levels of neuron-derived octopamine act on astrocytes via a trace amine-associated receptor 1-Orai1-Ca²⁺-calcineurin-mediated signaling pathway to stimulate lactate secretion. Lactate uptake in neurons via the monocarboxylase transporter 2-calcineurin-dependent pathway increases ATP and prevents neurodegeneration. Pathological increases of octopamine caused by α-synuclein halt lactate production in astrocytes and short-circuits the metabolic communication to neurons. Our work provides a unique function of octopamine as a modulator of astrocyte metabolism and subsequent neuroprotection with implications to α-synucleinopathies.

Keywords: astrocytes; calcineurin; lactate; octopamine; synuclein.

Fig. 1.

Partial inhibition of calcineurin induces neuroprotection by promoting lactate synthesis, which prevents α-syn toxicity in neurons and astrocytes. (A) Representative western blot for α-syn, phosphoserine-129 α-syn, and calcineurin from cerebral cortex lysates of CaMKII–Cre (control) and CaMKII–Cre–α-syn (α-syn) animals injected twice with FK506 or vehicle (Dimethyl Sulfoxide or DMSO) 4 d apart and killed at day six. Actin serves as loading control. (B) Densitometry quantitation from the WB in A of p-S129 α-syn/α-syn/actin. Data are normalized to control cultures treated with vehicle (DMSO). N = 3; **P < 0.01; ****P < 0.0001; one-way ANOVA, post hoc Tukey test. (C) Representative immunohistochemistry for GFAP of matched sections from cerebral cortex of animals in A. (Scale bar is 20 µm.) (D) Quantification of GFAP fluorescence intensity (F.I) and (E) GFAP-positive nuclei from animals in A N ≥ 5; n.s =nonsignificant; *P < 0.05; one-way ANOVA, post hoc Tukey test. (F) Lactate metabolite from rat primary cortical neuron–astrocyte cocultures transduced with either control lentivirus or α-synA53T driven by the synapsin promoter and treated with either vehicle (DMSO) or subsaturating (0.2 µM) doses of FK506 and processed for 5 to 6 wk post transduction. Representative data from 3 independent experiments run in duplicates; *P < 0.05; one-way ANOVA, post hoc Tukey test. (G) Lactate metabolite from cerebral cortex of control and α-syn transgenic animals in A treated with FK506, N ≥ 5; *P < 0.05; one-way ANOVA, post hoc Tukey test. (H) ATP content from rat primary cortical neuron–astrocyte cocultures described in (F) treated with exogenous lactate, FK506, or both 5 to 6 wk post transduction. Raw values were normalized to control—DMSO. N = 6 to 8; *P < 0.05; unpaired t test with Bonferroni correction. (I) Representative immunofluorescence image for MAP2+ and GFAP+ from rat primary cortical neuron–astrocyte cocultures in (H); (scale bar, 50 μM.) (J) GFAP-positive fluorescence intensity (F.I)/nuclei from cortical neuron–astrocyte cocultures in (H) normalized to control—DMSO-treated conditions. N = 3; 300 to 600 cells/biological replicate; ***P < 0.001; one-way ANOVA, post hoc Tukey test. (K) Number of MAP2-positive neurons normalized to control—DMSO-treated conditions described in H. N = 6 to 8; ***P < 0.001, one-way ANOVA, post hoc Tukey test.

Fig. 2.

MCT2 expression regulated by calcineurin mediates lactate neuronal survival and astrogliosis in α-syn-expressing cultures. (A) Representative immunohistochemistry for MCT2 from cerebral cortex of matched sections of CaMKII–Cre (control) and CaMKII–Cre–α-syn (α-syn) animals injected twice with FK506 or vehicle (DMSO) 4 d apart and killed at day six. [Scale bar is 50 µm (40×).] (B) Quantification of average MCT2 fluorescence intensity (F.I) per cell from A. N ≥ 5; **P < 0.01; one-way ANOVA, post hoc Tukey test. Data were normalized to control vehicle (DMSO). (C and D) Representative immunofluorescence for MAP2+ (C) and for GFAP+ (D) from rat primary cortical neuron–astrocyte cocultures cotransduced with either control lentivirus or α-synA53T driven by the synapsin promoter and a doxycycline-inducible shRNA for MCT2 or shRNA scrambled sequence as control. All cultures were treated with either vehicle (DMSO), subsaturating doses of FK506 (0.2 µM), lactate (1 mM), or combination of both lactate and FK506 once a week for 5 wk post transduction. (Scale bar, 50 μM.) These cultures were quantified for number of MAP2+ cells for neurons (E), ATP content (F) and GFAP fluorescence intensity (F.I)/nuclei (G). Data were normalized to control-ShCtrl-DMSO condition. N = 4 to 6; *P < 0.05; **P < 0.01; ****P < 0.0001; unpaired t test with Bonferroni correction. (H) Representative immunohistochemistry for MCT2 in the superior frontal cortex area in human DLB and PD/DLB cases and age-matched controls. An average of 5 sections from 5 control and 6 disease cases were analyzed. [Scale bar is 50 µm (40×).] (I) MCT2 intensity was measured per cell from H. N = 5 to 6; **P < 0.01; one-way ANOVA, post hoc Dunnett’s multiple comparison test.

Fig. 3.

Octopamine is a calcineurin-dependent metabolite released under α-syn proteotoxicity that mediates Ca²⁺ influx in astrocytes. (A) Metabolite screen from supernatants of rat primary cortical neuron–astrocyte cocultures transduced with either control lentivirus or α-synA53T driven by the synapsin promoter and treated with either vehicle (DMSO) or subsaturating (0.2 µM) doses of FK506 5 to 6 wk posttransduction. Metabolites that were significantly different (P < 0.05) between control and α-syn cultures were enriched for catecholamine synthesis by metabolite set enrichment analysis from MetaboAnalyst (McGill University). From these, only 3 hits were significantly different between α-syn and α-syn + FK506: octopamine, tryptamine, and DOPET. Representative data from 3 independent experiments run in duplicates; *P < 0.05; one-way ANOVA, post hoc Dunnett’s multiple comparison test. (B) Ca²⁺ imaging of primary rat cortical astrocyte-only cultures treated with a range of octopamine concentrations (0 to 10 µM). N = 3; 7 to 10 cells/biological replicate. (C) Representative single-cell Ca²⁺ imaging traces of primary rat cortical astrocyte-only cultures treated with 100 nM octopamine (N = 6). (D) Ca²⁺ imaging of primary rat cortical astrocyte-only cultures cotreated with octopamine (100 nM) and the TAAR1 receptor inhibitor, EPPTB (500 nM), washed, and then rechallenged with octopamine (100 nM). N = 3; 7 to 10 cells/biological replicate. (E) Ca²⁺ imaging of wild type (WT) or Orai1-KO (Orai1_fl/fl, GFAP-Cre) primary mouse cortical astrocyte-only cultures treated with octopamine (100 nM). N = 3; 7 to 10 cells/biological replicate. (F) Ca²⁺ influx rates of WT and Orai1-KO (Orai1_fl/fl, GFAP-Cre) transients from E. N = 3; 7 to 10 cells/biological replicate. (G) Ca²⁺ imaging of WT or Orai1-KO (Orai1_fl/fl, GFAP-Cre) primary mouse hippocampal astrocyte-only cultures treated with octopamine (100 nM) in the absence of extracellular Ca²⁺ and in 2mM Ca²⁺. N = 3; 7 to 10 cells/biological replicate.

Fig. 4.

Octopamine mediates lactate production in astrocytes in a TAAR1–Orai1–Ca²⁺–calcineurin-dependent manner. (A) Lactate measurements from WT and Orai1 KO (Orai1_fl/fl, GFAP-Cre) primary cortical astrocyte-only cultures treated with a range of octopamine concentrations (0.5 to 500 nM) over time. N = 9; *P < 0.05, one-way ANOVA, post hoc Tukey test. (B) Lactate measurements from WT and Orai1 KO (Orai1_fl/fl, GFAP Cre) primary cortical astrocyte-only cultures pretreated for 30 min with either FK506 (1 µM) or EPPTB (200 nM) and subsequently challenged with octopamine (100 nM) for 4 h. N = 3. *P < 0.05; one-way ANOVA, post hoc Dunnett’s multiple comparison test. (C) Heat map from the union of differentially expressed genes by RNA-Seq from primary cortical astrocyte-only cultures treated with vehicle, octopamine (100 nM), or octopamine (100 nM) + FK506 (1 µM). All data are relative to the vehicle treated (DMSO). Log10(P-value): Group 1= −14.5, Group 2= −10.1, Group 3= −8.5, Group 3= −28.2. (D) Measurements of generated lactate from ex vivo brain slices extracted from WT and Orai1_{fl/fl, GFAP-Cre} mice. After 3 d in culture, slices were treated with either 1× PBS or 100 nM octopamine. Samples of the media were taken at 4 h, 8 h, and 24 h post treatment. Normalized measurements were presented as fold change from baseline value measurements taken from vehicle-treated slices. N = 20 slices, N = 4 mice. (E) Measurements of rate of lactate production as calculated by the flux of lactate concentration between timepoints from D. (F) Measurements of the rate of lactate production with spatial separation from D. Slices were binned by stereotaxic coordinates lateral to bregma to show relative rates of lactate production in both spatial and temporal dimensions. (G) Representative immunohistochemistry from hippocampus CA3 for GFAP and DAPI of ex vivo brain slices from (D) 24 h posttreatment with either vehicle (Phosphate Buffered Saline or PBS) or 100 nM octopamine. (Scale bar, 50 µm.) (H) Quantification of GFAP fluorescence relative to the DAPI fluorescence as a measure of astrocyte reactivity from G. N = 3. **P < 0.01, ****P < 0.0001; one-way ANOVA, post hoc Dunnett’s multiple comparison test. (I) Representative immunofluorescence from prefrontal cortex for GFAP and DAPI of ex vivo brain slices from (D) 24 h posttreatment with either vehicle (PBS) or 100 nM octopamine. (J) Quantification of GFAP fluorescence relative to the DAPI fluorescence from I as a measure of astrocyte reactivity. N = 3. ****P < 0.0001; one-way ANOVA, post hoc Dunnett’s multiple comparison test. (Scale bar, 50 µm.)