Alterations of Dopaminergic Synapse and Mitochondrial Structure by Parkinson’s Disease Toxins

Research Article

J Mol Biol & Mol Imaging. 2014;1(2): 6.

Alterations of Dopaminergic Synapse and Mitochondrial Structure by Parkinson’s Disease Toxins

Meelim J Lee, Robert A Colvin2 and Daewoo Lee2*

1Athens High School, The Plains, OH, USA

2Neuroscience Program and Interdisciplinary Graduate Program in Molecular & Cellular Biology, Department of Biological Sciences, Ohio University, Athens, OH, USA

*Corresponding author: Daewoo Lee, Neuroscience Program and Interdisciplinary Graduate Program in Molecular & Cellular Biology, Department of Biological Sciences, Ohio University, Athens, OH, USA

§Current address Department of Bioengineering, Stanford University, Stanford, CA, USA

Received: October 02, 2014; Accepted: November 15, 2014; Published: November 23, 2014


Immunostaining with anti-tyrosine hydroxylase (TH) revealed that a small subset (~2.5%) of primary neurons (from rat embryonic E13-14 midbrains) is dopaminergic (DA). In this study, we tested whether DA neurons in culture are selectively degenerated by well-known PD toxins such as rotenone (5-100nM) and MPP+ (10µM). Both toxins significantly decreased the number of DA neurons and neurite length after 24-hour incubation. Interestingly, our results showed that mitochondria in DA neurites were also degenerated by these toxins. Since mitochondria play a critical role in proper synapse signaling and PD is directly linked to mitochondria dysfunction, we wanted to study altered properties of DA synaptic mitochondria in the early stage of PD. With triple staining of anti- TH, a fixable synaptic marker FM1-43fx and a mitochondrial dye MitoTracker Orange (MTO), we identified DA synaptic mitochondria. Our data demonstrated that DA synaptic mitochondria are degenerated by MPP+ and rotenone, and that this effect likely occurs prior to the degeneration of DA cell bodies. Therefore, our cellular PD model can be used to study presymptomatic alterations underlying development of PD pathology. Our study will help to understand mechanisms underlying selective loss of DA neurons, which is possibly due to the degeneration of DA synaptic mitochondria.

Keywords: primary dopamine neurons; MPP+; Rotenone; Dopamine synapse; Mitochondria


DA: Dopaminergic; DIV: Day(s) in vitro; FM1-43fx: A Fixable Synaptic Marker; FOV: Field Of View; MPP+: 1-Methyl-4- PhenylPyridinium; MTO: MitoTracker Orange; NB: Neurobasal; PD: Parkinson’s disease; TH: Tyrosine Hydroxylase


Dopamine (DA) is an important neurotransmitter mediating a variety of higher brain function (e.g. cognition, motor function; [1]). Indeed, selective loss of DA neurons in the substantia nigra is a key pathological feature of Parkinson’s disease (PD) which causes DA deficits and thus disturbs motor function [2, 3, 4]. However, preceding the selective loss of DA neurons, there must be changes in the sub-cellular structure of these neurons. It is known that neurite or synaptic loss precedes degeneration of DA cell bodies [5], strongly indicating that there is substantial damage to DA neurons and synapses at the sub-cellular level before PD symptoms are clinically detected. Therefore, characterization of these early changes is crucial not only to understand mechanisms underlying initiation of DA degeneration but also to provide clues on how PD pathology initiates and develops. Additionally, it contributes to development of early detection methods and therapeutic strategies for PD. However, the presymptomatic characteristics are not currently well understood at molecular or cellular levels.

Recent genetic discoveries firmly established that familial forms of PD are directly associated with mutations of certain genes [3, 6]. So far, at least 6 genes (a-Syn, parkin, UCHL-1, DJ-1, PINK1, LRRK2) have been identified and they are directly associated with the pathogenesis of PD [7, 8, 3]. In contrast, the majority of PD cases (~90 - 95%) are thought to occur sporadically without clear genetic linkage. Thus, neurotoxin-induced loss of DA neurons is widely used to model non-genetic, environmental onset of PD [9, 3].

The neurotoxins 1-methyl-4-phenylpyridinium (MPP+) and rotenone have been used in a wide range of animal PD models (e.g. worm, Drosophila, rat, primate) since they show selective loss of DA neurons and motor impairment. Therefore, we believe these two toxins to be excellent chemical tools for PD research examining early changes in sub-cellular structure of DA neurons. In this study, we examined early degenerative changes in sub-cellular structure (e.g. synapse, neurite length, synaptic mitochondria) of DA neurons mediated by PD toxins.

Materials and Methods

Rat DA neuronal cultures and PD toxin treatment

Primary neuronal cultures were prepared from embryonic rat ventral midbrain (E13-14) and grown in neurobasal (NB) culture media supplemented with B-27 as previously described [10, 11]. Timed pregnant Sprague Dawley rats were ordered from a commercial supplier (Harlan Laboratories). Primary neuronal cultures were maintained in 37°C incubator with 5% CO2 supply. 6-8 day old neurons were treated with PD toxins, MPP+ (10µM) or rotenone (5, 20 and 100nM) for 24 hours. Finally, cultured neurons were fixed and stained with antibodies (e.g. anti-TH) and/or fluorescent dyes (e.g. FM1-43fx, MitoTracker Orange - see below). PD toxins were purchased from Sigma and all other reagents used here were purchased from Invitrogen. For all our experiments, MPP+ was prepared and disposed according to the guideline reviewed in [12]. Rotenone was similarly handled.

Quantification of DA neurites

Anti-TH antibody (Chemicon) was used to identify DA neurites in addition to the cell body. Stained neurites were observed under a fluorescent microscope (Olympus IX71). Images were taken using a Spot CCD digital camera (Diagnostic Instruments). Using a neuron/ neurite tracing software Neurolucida (BMF software), we manually traced neurites and cell bodies to create “skeletons,” which allowed the Neurolucida software to calculate neurite length (Figure 2B).

Quantification of DA mitochondria

DA mitochondria were stained using fixable mitochondriaspecific fluorescent dyes (i.e. MitoTracker Orange (MTO), Invitrogen). Cultures were incubated in 50nM MTO for 1 hour at 37°C in NB media. MTO labels all mitochondria in neuronal cultures; thus signals positive for both anti-TH and MTO staining were considered DA mitochondria and thus quantified using a Javabased image processing software program ImageJ. Morphological properties of DA mitochondria (e.g., size, number, elongation, etc) were examined as described previously [13].

Quantification of DA synapses

FM1-43fx is a fixable styryl dye known to identify functional synapses, particularly presynaptic terminals. This lipophilic dye (50nM) is taken-up by presynaptic endocytosis after neurons are depolarized by exposure to 50mM KCl for 5 min [14]. Since this dye recognizes all functional synapses, rat neuronal cultures were subsequently stained with anti-TH antibody. Regions double-stained with FM1-43fx (a fixable form of FM1-43, Invitrogen) and anti-TH antibody were identified as functional DA synapses (presynaptic punctae).

DA synaptic mitochondria

Triple staining was required in order to identify mitochondria specific to DA synapses. Therefore, DA neuronal cultures were stained with anti-TH, FM1-43fx and MTO. All three signals (i.e. FM1- 43fx, MTO, anti-TH) were distinguishable using epifluorescence microscopy. The overlapping fluorescent signals were considered DA synaptic mitochondria and quantified using ImageJ.


Dopaminergic (DA) neurodgeneration by PD toxins

Using the primary rat midbrain neuronal cultures containing dopaminergic (DA) neurons (Figure 1), we first examined whether DA neurons were degenerated by exposure to PD toxins (MPP+ and rotenone). DA neurons were identified using a DA marker antityrosine hydroxylase (TH) antibody. The percent of DA neurons in the culture was 2.5+/-2.4% (n=18). This percent was significantly reduced when primary neuronal cultures (6-8 days old) were incubated for 24 hours with either 10µM MPP+ or 100nM rotenone (Figure 1D). The effect of 20nM rotenone was intermediate while there was no change in the number of DA neurons when exposed to 5nM rotenone (Figure 1D). In order to examine whether DA neurons are selectively degenerated, we determined the total number of neurons in each field of view (FOV) in the presence or absence of toxin exposure. Individual cells in the neuronal culture were quantified using a nuclear dye DAPI [15, 16]. Figure 1E shows that there was no observable difference in the total number of cells in culture with or without exposure, confirming the selective degeneration of DA neurons by these two PD toxins. Our results demonstrate that we successfully developed a cellular PD model using these toxins. Therefore, this model was used to further study early sub-cellular changes in DA neurons.