Dopamine neurons exhibit emergent glutamatergic identity in Parkinson's disease

Abstract

Loss of midbrain dopamine neurons causes the cardinal symptoms of Parkinson's disease. However, not all dopamine neurons are equally vulnerable and a better understanding of the cell-type specific properties relating to selective dopamine neuron degeneration is needed. Most midbrain dopamine neurons express the vesicular glutamate transporter VGLUT2 during development and a subset continue to express low levels of VGLUT2 in adulthood, enabling the co-release of glutamate. Moreover, VGLUT2 expression in dopamine neurons can be neuroprotective since its genetic disruption was shown to sensitize dopamine neurons to neurotoxins. Here, we show that in response to toxic insult, and in two distinct models of alpha-synuclein stress, VGLUT2 dopamine neurons were resilient to degeneration. Dopamine neurons expressing VGLUT2 were enriched whether or not insult induced dopamine neuron loss, suggesting that while VGLUT2 dopamine neurons are more resilient, VGLUT2 expression can also be transcriptionally upregulated by injury. Finally, we observed that VGLUT2 expression was enhanced in surviving dopamine neurons from post-mortem Parkinson's disease individuals. These data indicate that emergence of a glutamatergic identity in dopamine neurons may be part of a neuroprotective response in Parkinson's disease.

Keywords: Parkinson's disease; alpha-synuclein; dopamine; selective vulnerability; vesicular glutamate transporter 2.

Figure 1

Medial forebrain bundle 6-OHDA increases the fraction of DA neurons expressing VGLUT2 in both SNc and VTA. (A) Schematic of unilateral medial forebrain bundle (MFB) 6-OHDA injection leading to anterograde and retrograde degeneration of midbrain DA neurons. (B) Cartoon depicting selective 6-OHDA (red circles) uptake via the DA transporter (DAT, green) into DA varicosities where it induces rapid degeneration through oxidative stress and mitochondrial disruption. (C) Example images showing coronal sections with TH+ midbrain DA neurons from vehicle- and 6-OHDA-injected animals. Note reduction in TH mRNA signal in both VTA (blue arrows) and SNc (white arrows) of 6-OHDA treated mouse (right panel). (D) DA neuron counts are reduced, but the fraction of DA neurons that express VGLUT2 is increased in SNc and (E) in VTA; **P < 0.01; ***P < 0.001, two-tailed unpaired t-test.

Figure 2

VGLUT2 expression emerges in response to α-synuclein fibril pathology. (A) PFF of α-synuclein injected ipsilaterally into striatum leads to accumulation of α-synuclein aggregates in distal SNc DA neurons. (B) Purified recombinant α-synuclein fibrillized by sonication induces synuclein aggregation in vivo. (C) Three months after unilateral injection, co-labelling for TH mRNA and Ser-129 phosphorylated α-synuclein (pSyn) protein in coronal sections through SNc revealed reduced TH signal concomitant with pSyn labelling in PFF- but not vehicle-treated mice. (D) DA neuron counts in SNc are reduced, but the fraction of DA neurons expressing VGLUT2 is increased compared to vehicle controls, at both 3 months and (E) 6 months following PFF exposure; *P < 0.05; **P < 0.01, two-tailed unpaired t-test.

Figure 3

Emergence of VGLUT2 without DA neuron loss after heterologous expression of human α-synuclein^A53T. (A) Strategy for unilateral expression of AAV-DIO-human α-synuclein^A53T in the SNc of DAT^Cre mice. (B) AAV construct showing that in the presence of Cre recombinase, α-synuclein^A53T is recombined to allow for cell-type-specific expression. (C) Immunohistochemistry shows expression of GFP or human α-synuclein^A53T, detected in TH+ DA neurons in coronal sections through SNc using the human α-synuclein-specific antibody 15G7. (D) SNc DA neuron counts were not significantly altered 3 months after heterologous expression of α-synuclein^A53T (left), but the fraction of DA neurons expressing VGLUT2 was increased (right); **P < 0.01, two-tailed unpaired t-test.

Figure 4

VGLUT2+ DA neurons are enriched in SNc of human Parkinson’s disease. (A) Widefield view of transverse section containing SNc from control and Parkinson’s disease participants with chromogenic labelling for mRNAs encoding TH (diffuse magenta) and VGLUT2 (blue puncta) counter-stained with haematoxylin; brown signal is neuromelanin; cp = cerebral peduncle; RN = red nucleus. (B) Higher magnification images of SNc DA neurons expressing TH without VGLUT2 (left), expressing TH and VGLUT2 (middle), or RN neurons expressing VGLUT2 without TH (right). (B1–3) Inset images zoomed on boxes as depicted from B. (C) Reduced SNc DA neuron counts in sections from Parkinson’s disease patients; (D) but an increased fraction of SNc DA neurons expressed VGLUT2 in Parkinson’s disease compared to age-matched control participants not diagnosed with Parkinson’s disease; *P < 0.05; **P < 0.01, two-tailed unpaired t-test.