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. 2018 Feb 1;128(2):774-788.
doi: 10.1172/JCI95795. Epub 2018 Jan 16.

Role for VGLUT2 in Selective Vulnerability of Midbrain Dopamine Neurons

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Free PMC article

Role for VGLUT2 in Selective Vulnerability of Midbrain Dopamine Neurons

Thomas Steinkellner et al. J Clin Invest. .
Free PMC article

Abstract

Parkinson's disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. Here, we addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development.

Keywords: Mouse models; Neurodegeneration; Neuroscience; Parkinson’s disease.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. dVGLUT overexpression causes loss of select dopaminergic populations in Drosophila.
Drosophila VGLUT (dVGLUT) was expressed specifically in DA neurons via the TH-GAL4 expression driver and termed dVGLUT high expressors (dVGLUT HE). dVGLUT HE brains were compared with control brains expressing dVGLUT at WT levels. Presynaptic DA neurons in both dVGLUT HE and control flies were labeled with TH-GAL4–driven GFP and whole living brains visualized by multiphoton microscopy. (AC) dVGLUT HE brains demonstrated complete loss of DA innervation to the ellipsoid body (EB). Insets in A and B highlight the EB region. Two-way ANOVA; main effect of genotype: F(1,10) = 1,316, P < 0.0001; time × genotype interaction: F(3,30) = 26.5, P < 0.0001. (DF) While the overall morphology of the fan-shaped body (FSB) was preserved in dVGLUT HE brains, there were fewer DA cell bodies innervating it [2-way ANOVA; main effect of genotype: F(1,9) = 6.6, P = 0.03], and diminished axon length compared with controls [2-way ANOVA; main effect of genotype: F(1,4) = 277, P < 0.0001]. (G) The number of DA neurons innervating the subesophageal ganglion (SEG) showed no significant changes, but axon length in dVGLUT HE was significantly reduced compared with WT [2-way ANOVA; main effect of genotype: F(1,4) = 35.7, P = 0.004]. Comparable results were obtained from n ≥ 3 independent experiments. Images are projected Z series of coronal sections 3 days after eclosion. Scale bars: 50 μm; insets, 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001 across genotype, within time point by Sidak’s multiple-comparisons post hoc.
Figure 2
Figure 2. Heterologous expression of VGLUT2 in adult DA neurons enables glutamate release.
(A and B) AAV containing Vglut2 transgene in the double inverted open reading frame configuration (AAV-DIO-VGLUT2) (A) and strategy to selectively express VGLUT2 and ChR2:mCherry in Cre recombinase–expressing DA neurons of VGLUT2 cKO mice (B). (C) AAV-DIO-ChR2:mCherry and blue light pulses were used to evoke transmitter release from DA terminals. No excitatory currents were detected in VGLUT2 cKO mice (controls), but heterologous expression of VGLUT2 led to light-evoked AMPA-type EPSCs that were blocked by DNQX. Paired t test, t = 4.6, df = 4, n = 5; **P = 0.01. Scales: 100 pA, 50 ms. (D) Histology confirms expression of VGLUT2 and ChR2:mCherry in TH+ neurons, and shows reduced TH signal on the side of injection. Scale bar: 100 μm.
Figure 3
Figure 3. Cell loss following heterologous expression of VGLUT2 in adult SNc DA neurons.
DATCre or DAT+/+ mice were unilaterally injected with AAV-DIO-VGLUT2, AAV-DIO-GFP, or AAV-DIO-VMAT2:pHluorin into the SNc and euthanized at indicated time points, and coronal sections were immunostained against TH. (A) Images of sections through SNc and VTA show pronounced loss of DA neurons in the SNc on the side of injection, while medial VTA is relatively spared. Scale bar: 500 μm. (B) TH+ cell counts in the SNc assessed by unbiased stereology. (C) Striatal sections stained for TH after unilateral viral injection of GFP or VGLUT2 at indicated time points. Note that holes on the contralateral side were to track the uninjected hemisphere. Scale bar: 1,000 μm. (DF) Loss of TH signal in the striatum after VGLUT2 overexpression assessed by densitometry. Ten days: unpaired t test, t = 1.0, df = 4, P = 0.37, n = 3 per group; 21 days: unpaired t test, t = 3.090, df = 4, P = 0.037, n = 3 per group; 240 days: unpaired t test, t = 13.8, df = 6, P < 0.0001, n = 4 per group. See Table 1 for additional statistics. *P < 0.05, ****P < 0.0001.
Figure 4
Figure 4. Heterologous expression of VGLUT2 in DA neurons induces Parkinsonian behavior.
WT VGLUT2 or GFP (control) was unilaterally expressed in the SNc of DATCre mice, and mice were tested in the open field beginning 21 days after surgery. (A) VGLUT2 expression significantly reduced spontaneous locomotor activity [left panel: 2-way ANOVA followed by Sidak’s multiple comparisons; main effect of treatment: F(1,312) = 86, P < 0.0001; right panel: unpaired t test, t = 3.7, df = 26, P = 0.001; GFP n = 12, VGLUT2 n = 17]. (B and C) Heterologous VGLUT2 expression significantly decreased locomotion in response to amphetamine [B; left panel: 2-way ANOVA followed by Sidak’s multiple comparisons; main effect of treatment: F(1,972) = 233, P < 0.0001; right panel: unpaired t test, t = 2.6, df = 27, P = 0.01; GFP n = 13, VGLUT2 n = 16] or cocaine [C; left panel: 2-way ANOVA followed by Sidak’s multiple comparisons; main effect of treatment: F(1,1008) = 132, P < 0.0001; right panel: unpaired t test: t = 2.8, df = 28, P = 0.01; GFP n = 13, VGLUT2 n = 17]. Left panels show time courses in 5-minute bins; right panels are summated over 60 minutes. (D) Unilateral VGLUT2 expression led to ipsiversive rotational behavior [2-way ANOVA followed by Sidak’s multiple comparisons; main effect of treatment: F(1,56) = 25, P < 0.0001; GFP n = 13, VGLUT2 n = 17]. (E and F) Rotations were reversed to contraversive by apomorphine [E; 2-way ANOVA followed by Sidak’s multiple comparisons; main effect of treatment: F(1,56) = 4.5, P = 0.04; GFP n = 13, VGLUT2 n = 17] or l-DOPA/benserazide [F; 2-way ANOVA followed by Sidak’s multiple comparisons; main effect of treatment: F(1,56) = 11, P = 0.002; GFP n = 13, VGLUT2 n = 17]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5
Figure 5. VGLUT2 is transiently expressed in most mouse midbrain DA neurons.
(A and B) Coronal sections from mice expressing zsGreen reporter in a VGLUT2Cre driver line reveal marked colocalization with the DA maker TH in both the SNc (A) and the VTA (B). Scale bars: 100 μm. (C) Higher-magnification image of SNc DA neurons labeled with zsGreen. Scale bar: 20 μm. (D) Fraction of TH-labeled cells that express zsGreen (n = 2).
Figure 6
Figure 6. Endogenous VGLUT2 expression emerges in SNc of surviving DA neurons after neurotoxic insult.
(A) SNc images labeled using probes against Th and Vglut2 mRNA following the unilateral injection of 6-OHDA (right panel) or vehicle (left panel) into the dorsal striatum. Scale bars: 500 μm. (B) Higher-magnification images of injected (right panel) and uninjected (left panel) SNc neurons after 6-OHDA. Arrows indicate Th/Vglut2 colabeling. Scale bars: 10 μm. (C) Quantification of Th+ cells on ipsi- and contralateral sides in the SNc after vehicle or 6-OHDA treatment [left panel: 2-way ANOVA followed by Sidak’s multiple comparisons; treatment × hemisphere effect: F(1,16) = 6.4, P < 0.05; treatment effect: F(1,16) = 8.9, P < 0.01; vehicle n = 4, 6-OHDA n = 6] and fraction of Th-labeled cells that colabel for Vglut2 [right panel: 2-way ANOVA followed by Sidak’s multiple comparisons; treatment × hemisphere effect: F(1,16) = 17.2, P < 0.001; treatment effect: F(1,16) = 20.4, P < 0.001; vehicle n = 4, 6-OHDA n = 6]. (D) Quantification of Th+ cells on ipsi- and contralateral sides in the VTA after vehicle or 6-OHDA treatment (left panel: unpaired t test; t = 1.2, df = 6, n = 4 per group, P > 0.05) and fraction of Th-labeled cells that colabel for Vglut2 (right panel: unpaired t test; t = 1.1, df = 6, n = 4 per group, P > 0.05). (E) Histogram showing the distribution of Vglut2+ puncta in Th+ cells in the SNc after 6-OHDA (red) or vehicle (black) treatment. (F) Cumulative probability blot comparing 6-OHDA–induced increase in the number of Vglut2+ puncta per TH+ cell in the SNc (vehicle n = 4, 6-OHDA n = 6; Kolmogorov-Smirnov [KS] test). *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 7
Figure 7. Conditional knockout of VGLUT2 makes DA neurons more susceptible to 6-OHDA and MPTP.
(A) Mice received intrastriatal injections with 6-OHDA (4 μg) or vehicle unilaterally into the dorsal striatum. (B) Sections through the SNc were stained for TH and TH+ SNc neurons counted with unbiased stereology [treatment effect: F(1,18) = 108, P < 0.0001; vehicle n = 4, 6-OHDA n = 6–8]. (C) Mice were systemically treated with MPTP or vehicle according to an acute regimen (4 injections in 1 day with 15 mg/kg i.p.) and (D) TH+ cells were counted by unbiased stereology [treatment effect: F(1,10) = 14.8, P < 0.01; n = 3–4]. (E) Mice were systemically treated with MPTP or vehicle according to a chronic regimen (30 mg/kg daily over 5 days, i.p.) and (F) TH+ cells were counted by unbiased stereology [treatment effect: F(1,8) = 7.8, P < 0.05; n = 3]. Scale bars: 500 μm. *P < 0.05, ****P < 0.0001; Sidak’s multiple comparisons.

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