Regional microglia are transcriptionally distinct but similarly exacerbate neurodegeneration in a culture model of Parkinson’s disease

All animals used in this study were maintained in accordance with the Office of Animal Resources at Thomas Jefferson University. The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Thomas Jefferson University, protocol #457K/01499.

Timed pregnancies were performed using male and female rats from our colony of animals expressing green fluorescent protein under control of the human tyrosine hydroxylase promotor (TH-GFP) [1]. Embryonic day zero (E0) was determined as the first day a vaginal plug was visible. On E14.5, embryos were harvested and placed in ice cold DPBS (Gibco #14190-144; no CaCl2 or MgCl2). GFP+ pups were visualized under a dissection microscope (Nikon SMZ1500; adjustable × 1–× 11.5 objective) by using a high-pressure mercury lamp with a 495-nm filter attachment. The midbrain was surgically extracted and cleaned of non-GFP+ tissues, and the VTA and SN were carefully separated. Tissues were collected and placed in 2.5 mL of enzymatic dissociation solution containing 5 mg DNAse I (Sigma #10104159001), 5 mg Papain (Sigma #10108014001), and 50 mg l-cysteine (Sigma) for up to 30 min. Solution was gently agitated every 5 min to aid in dissociation. NEP basal media (DMEM/F12, B-27 and N2 Supplements (Gibco), 1 mg/mL BSA, and Penn/Strep) was added to terminate the enzymatic reaction. Tissues were then briefly spun at 1000 rpm for 2 min, and the supernatant media was aspirated. Tissues were re-suspended in culture media (NEP basal media + 5% FBS) for 5 min prior to mechanical dissociation. For mechanical dissociation, a 1-mL pippetor was used with low retention tips and tissues were gently triturated eight to ten times. Cells were then pelleted for 5 min at 1000 rpm, re-suspended in culture media, and counted on an automated hemocytometer (Countess FLII Invitrogen). Cells were plated in a microdrop on 96-well tissue culture plates or 8-well chamber slides pre-coated with poly-d-lysine (0.5 mg/mL, overnight at 37 °C) at a density of 5–7 × 10⁴ cells in approximately 20 μL of culture media. Cells were allowed to attach to the substrate before media was added to final volume of 100–200 μL per well. Cells were allowed to mature for 6 days prior to the addition of isolated microglia (see below) in a 1:1 microglia to neuron ratio and allowed to mature for 24 h prior to MPP⁺ treatment. This ratio was established based on previous studies demonstrating similar numbers of MAC1+ or Iba1+ microglia and TH+ neurons within the SN [16, 28]. Experiments where inhibition of microglia was performed utilized the commercially available TLR4 antagonist 2-acetamidopyranoside (TLR4-IN-C34, Sigma Cat# SML0832). On day 7 in vitro (DIV7), C34 was added to cultures 2 h prior to MPP⁺ addition. Cultures were fixed 24–48 h after MPP⁺ treatment, and the full well was imaged using a Nikon Eclipse TI-e fitted with a Photometrics Coolsnap ES2 camera. Images were analyzed in ImageJ using a custom-designed macro for cell quantification. TH-GFP+ cell counts were compared to appropriate non-treated controls.

Astrocytes and microglia were collected using previously published methods [2‐4]. Briefly, postnatal day 1–5 GFP+ pups were anesthetized on ice and quickly decapitated. Brains were removed and placed in ice cold DPBS (Gibco). GFP+ brains were visualized under a dissection microscope (Nikon SMZ1500). The midbrain and cortices were carefully dissected away from other brain regions and non-GFP+ tissues of the midbrain were cut away. The SN and VTA regions were carefully separated and collected in ice cold PBS. Whole cortices were collected and dissected into smaller pieces prior to enzymatic digestion. Tissues were then subjected to enzymatic digestion using a trypsin (0.1%)/DNAse I (0.5 mg/mL) mixture for 5 min at 37 °C. The supernatant enzyme was aspirated carefully, and tissues were washed twice in DPBS. Tissues were then mechanically dissociated in 0.5 mg/mL DNAse I and gently triturated eight to ten times. Cells were then pelleted for 5 min at 1000 rpm, re-suspended in astrocyte media (DMEM/F12 + 10% FBS and Penn/Strep) and plated on tissue culture flasks. Cultures were maintained for 2–4 weeks with media changes every other day. To separate astrocytes (adherent) and microglia (non-adherent) for experimental use, flasks were shaken at 220 RPM for 24 h and the non-adherent cells collected. The cell suspension was passed through a 70-mm cell strainer prior to centrifugation at 1000 rpm for 5 min. The cells were re-suspended in culture media for experimental use. Samples of non-adherent cells were stained for the microglial markers Iba1 (allograft inflammatory factor 1 (Iba1, Wako #019-19741, RRID:AB_2665520) and CD11b (BD Biosciences Cat# 550299, RRID:AB_393594) to confirm homogeneity of the culture.

Neuron-astrocyte co-cultures were prepared similarly to previously published methods [5]. Briefly, isolated regional astrocytes were trypsinized, dissociated to a single cell suspension, and quantified. Increasing numbers of astrocytes (60,000–120,000) were plated onto 48-well tissue culture plates. Cells were allowed to adhere and grow for 48 h prior to further experimentation. Subsequently, embryonic neuron preparations were carried out to obtain TH-GFP+ neurons. Neurons were plated onto regional astrocytes in a homo- and heterotypic manner at 2:1 and 4:1 (astrocyte to neuron) ratios. These ratios were established based on previous studies examining the number of astrocytes in the midbrain [28, 29], as well as our previous work demonstrating increased ratios of VTA but not SN astrocytes mediate protection from MPP⁺ toxicity (Kostuk et al. 2018, in submission). Twenty-four hours after plating, all cultures were treated with 1 μM AraC to halt astrocytic proliferation. Cultures were then allowed to mature for five more days in vitro prior to microglial addition. Isolation and collection of SN and VTA microglia in sufficient numbers for experimental manipulation would require a vast number of animals to be sacrificed; as such, cortical microglia were used as they were easier to obtain in sufficient numbers. Microglia were added in a 1:1 and 2:1 (microglia to neuron) ratio. Twenty-four hours after microglial addition, MPP⁺ was added at 50 μM for 24–48 h. After MPP⁺ treatment, all cells were fixed and GFP+ cells were quantified using the Nikon Eclipse camera system described above.

Total RNA was isolated directly from freshly collected cells in TRIzol (Invitrogen), a modification of the guanidine isothiocyanate-phenol-chloroform extraction method. cDNA was synthesized by using at least 1 μg total RNA in a 20-μl reaction with Superscript IV (Invitrogen) and oligo (dT)12–18 (Invitrogen). One microliter of RNase H (Invitrogen) was added to each reaction tube, and the tubes were incubated for 20 min at 37 °C before proceeding to real-time PCR.

Real-time PCR was carried out on the 7500 Real Time PCR System using SYBR green PCR master mix (both from Applied Biosystems). GAPDH was used as an internal control. PCR analyses were conducted in triplicate for each sample. The reaction mix consisted of 6.35 ng cDNA, 0.5 μM forward and reverse primer mix, and × 1 SYBR green PCR master mix. Reactions were run according to manufacturer protocols for at least 40 cycles. Data were analyzed using the ratiometric ΔΔCT method, and the mean relative mRNA expression for each sample was reported. All primer sequences used can be found in Additional file 1: Table S1.

To examine the potential regionality of microglia within the midbrain, we isolated microglia from SN and VTA and compared them to microglia from cortex (CTX) using postnatal day 1–5 transgenic rats. Microglial purity was estimated at 95–100% as the vast majority of cells stained for the microglial markers Iba1 (Fig. 1a) and CD11b (Fig. 1b). Regionally isolated microglia were then collected and examined for their basal cytokine profile. We found that regionally isolated microglia exhibit subtle differences in cytokine expression profiles (Fig. 1c, d). In comparison to isolated CTX microglia (Fig. 1c, left column), SN microglia exhibit greater levels of the pro-inflammatory cytokines IL3, CCL3, and CCL4, while exhibiting diminished expression of IFNγ. VTA microglia (Fig. 1c, right column) conversely express greater levels of pro-inflammatory RANTES, Cxcl12, and CCL19, with decreased expression of IL10. An additional comparison was performed with CTX and SN microglia against isolated VTA microglia (Fig. 1d). We found that both SN and CTX have greatly upregulated levels of IL10 and diminished expression of CCL19 (Fig. 1d). Together, these results demonstrate a diverse regionality to microglia of the midbrain based on their baseline cytokine expression levels.

Previous studies have demonstrated that in vivo treatment with the PD toxin MPTP causes microglial activation [16, 30]. Therefore, we examined the response of our regionally isolated microglia to the active metabolite of MPTP, MPP⁺, to determine whether regionality results in differential activation profiles. We found that SN microglia (Fig. 2, left column) strongly upregulate the expression of pro-inflammatory IL1β, IL12α, CCL3, CCL4, and CCL19, while decreasing expression of anti-inflammatory IFNγ in response to toxin treatment. Interestingly, while there are some similarities in the pro-inflammatory response of VTA microglia to MPP⁺ (Fig. 2, middle column), such as upregulation of IL1β, CCL3, and CCL4, these microglia differ in their strong downregulation of Cxcl12 and significant upregulation of IL3. Finally, CTX microglia differ greatly from the other two regions examined (Fig. 2, right column). Their upregulation of pro-inflammatory cytokines is limited to TNFα and RANTES, but with strongly decreased expression of both IL1β and IL12α. However, CTX microglia mimic SN in their downregulation of anti-inflammatory IFNγ in response to MPP⁺ and also decreased expression of IL6. The differential expression of cytokines in response to MPP⁺ treatment would suggest that the addition of regionally isolated microglia would result in differential exacerbation of MPP⁺ toxicity of DA neurons.

Due to the differences seen in the cytokine profile of regionally isolated microglia, both at baseline and in response to MPP⁺, we hypothesized that regionally isolated microglia would cause differential exacerbation of MPP⁺ toxicity of midbrain DA neurons grown with their astrocytes. We find that compared to control cultures (M = 100, SEM = 9.3), the addition of regionally isolated microglia (SN light green, M = 105, SEM = 4.1; VTA tan, M = 102, SEM = 3.1; and CTX light purple bars, M = 105, SEM = 3.2) do not cause DA neuron degeneration in either SN (Fig. 3a) or VTA (data not shown, one-way ANOVA, F (7, 16) = 1.313 p = 0.3064) neuron to astrocyte cultures. Interestingly, despite the regionally associated expression differences of pro-inflammatory cytokines upon MPP⁺ stimulation, all regionally isolated microglia similarly exacerbate MPP⁺ toxicity of SN DA neurons (Fig. 3a, SN dark green, M = 27, SEM = 1.5; VTA orange, M = 26, SEM = 2.1; and CTX dark purple bars, M = 23, SEM = 1.8; one-way ANOVA with Tukey’s post hoc multiple comparisons test, F (7, 16) = 84.2, *** = p < 0.001), whereas VTA DA neurons (data not shown) are robustly protected from the effect of microglia in neuron to astrocyte cultures. These results suggest that VTA, but not SN, astrocytes may be protective of their regional DA neurons despite the addition of damaging microglia regardless of their region of origin.

To confirm that microglial released factors are indeed responsible for the exacerbation of MPP⁺ toxicity, we sought to prevent microglial activation by using a recently developed, commercially available TLR4 antagonist TLR4-IN-C34 (C34) [31]. When C34 is applied to cultures 2 h prior to MPP⁺ administration, we found that C34 (Fig. 3b, light purple bar, M = 98, SEM = 1.7) itself has no effect on the health of mixed cultures of SN neurons, astrocytes, and microglia (control, blue bar, M = 96, SEM = 3.7). However, coupled with MPP⁺ treatment (Fig. 3b, dark purple, M = 56, SEM = 1.4), we demonstrated significantly decreased exacerbation of MPP⁺ toxicity (red bar, M = 35, SEM = 2.1) of SN DA neurons (one-way ANOVA with Tukey’s post hoc multiple comparisons test, F (3, 14) = 144.4, **** = p < 0.0001). Cultures where C34 inhibition has occurred resemble our previously established data where microglia are absent from cultures (Kostuk et al. 2018, in submission). This further supports that factors released from microglia indeed exacerbate MPP⁺ toxicity of these neurons.

As we demonstrated that VTA neurons are robustly protected from MPP⁺ toxicity despite the presence of microglia, we sought to determine whether this protection is due to astrocytic released factors. We created both homo- and heterotypic astrocyte-neuron cultures from the SN and VTA region in varying ratios to determine if, similar to our previous findings, greater numbers of astrocytes would afford protection despite the presence of microglia. We found that in both homotypic (SN DA neurons on SN astrocytes, Fig. 4, solid blue bars: 1:2:1 ratio M = 37, SEM = 3.4; 1:4:1 ratio M = 34, SEM 1.5) and heterotypic (VTA DA neurons on SN astrocytes, Fig. 4, solid red bars: 1:2:1 ratio M = 45, SEM = 2.1; 1:4:1 ratio M = 41, SEM = 1.2) co-cultures, higher ratios of SN astrocytes were incapable of affording greater protection of both SN and VTA neurons when microglia were present at a ratio equal to neurons. Conversely, VTA astrocytes exhibited ratio-dependent increases in protection of both SN and VTA DA neurons despite the presence of exacerbatory microglia in both homotypic (VTA DA neurons on VTA astrocytes, Fig. 4, hatched red bars: 1:2:1 ratio M = 82, SEM = 1.2; 1:4:1 ratio M = 96, SEM = 0.75) and heterotypic (SN DA neurons on VTA astrocytes, Fig. 4, hatched blue bars: 1:2:1 ratio M = 86, SEM = 1.2; 1:4:1 ratio M = 89, SEM = 1.8). To further our understanding of the protection provided by VTA astrocytes, we then sought to tip the balance towards the deleterious effects of microglia by increasing their proportion of the cells in the dish. We found that the addition of microglia at a 2:1 ratio to neurons in the presence of the most protective astrocyte ratio (4:1 astrocytes to neurons) does result in DA neuron death simply because more microglia were added (data not shown). However, when coupled with MPP⁺ treatment, we found that the protective effect of VTA astrocytes was significantly diminished (Fig. 4; SN neurons blue vertically stripped bar, M = 57, SEM = 0.62 and VTA neurons red vertically stripped bar, M = 51, SEM = 1) by increasing the ratio of microglia (one-way ANOVA with Tukey’s post hoc multiple comparisons test; F (9, 28) = 270.8, ****p < 0.0001). These results further suggest that microglial released factors are able to overcome the protective effect of astrocytes.