Blocking soluble tumor necrosis factor signaling with dominant-negative tumor necrosis factor inhibitor attenuates loss of dopaminergic neurons in models of Parkinson's disease

Abstract

The mechanisms that trigger or contribute to loss of dopaminergic (DA) neurons in Parkinson's disease (PD) remain unclear and controversial. Elevated levels of tumor necrosis factor (TNF) in CSF and postmortem brains of PD patients and animal models of PD implicate this proinflammatory cytokine in the pathophysiology of the disease; but a role for TNF in mediating loss of DA neurons in PD has not been clearly demonstrated. Here, we report that neutralization of soluble TNF (solTNF) in vivo with the engineered dominant-negative TNF compound XENP345 (a PEGylated version of the TNF variant A145R/I97T) reduced by 50% the retrograde nigral degeneration induced by a striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA). XENP345 was neuroprotective only when infused into the nigra, not the striatum. XENP345/6-OHDA rats displayed attenuated amphetamine-induced rotational behavior, indicating preservation of striatal dopamine levels. Similar protective effects were observed with chronic in vivo coinfusion of XENP345 with bacterial lipopolysaccharide (LPS) into the substantia nigra, confirming a role for solTNF-dependent neuroinflammation in nigral degeneration. In embryonic rat midbrain neuron/glia cell cultures exposed to LPS, even delayed administration of XENP345 prevented selective degeneration of DA neurons despite sustained microglia activation and secretion of solTNF. XENP345 also attenuated 6-OHDA-induced DA neuron toxicity in vitro. Collectively, our data demonstrate a role for TNF in vitro and in vivo in two models of PD, and raise the possibility that delaying the progressive degeneration of the nigrostriatal pathway in humans is therapeutically feasible with agents capable of blocking solTNF in early stages of PD.

Figure 1.

Blocking TNF signaling in the nigra attenuates striatal 6-OHDA-induced loss of nigral DA neurons and drug-induced rotational behavior. A unilateral striatal lesion was induced by injecting 6-OHDA (20 μg) into the CPu complex of young adult rats; mock-lesioned animals received an injection of saline. Animals were stereotaxically implanted with an ipsilateral striatal or nigral indwelling cannula connected to an Alzet 2002 osmotic pump to deliver saline vehicle or XENP345 (0.08 mg · kg⁻¹ · d⁻¹) over a 2 week period. Animals were anesthetized, and brains were fixed for immunohistochemical analyses of TH/NeuN-IR neurons by intracardiac perfusion 3 weeks after the lesion. Panels from top to bottom represent increasing magnification of representative brain sections used to obtain stereological estimates of nigral DA neuron number in 6-OHDA-lesioned animals implanted with pump preloaded with saline [contralateral unlesioned side (a); lesioned/pump side (b)] or implanted with nigral pump preloaded with XENP345 [contralateral unlesioned side (c); lesioned/pump side (d)]. Scale bar: top panel, 100 μm; middle panel, 50 μm; bottom panel, 10 μm. e, Stereological estimate of DA neuron number (TH/NeuN-IR cells) in SNpc expressed as a percentage of the contralateral side (solid bars). Statistical significance was evaluated by ANOVA followed by post hoc comparison test between groups and to unlesioned control group. Values expressed are group mean + SEM; *p < 0.05; **p < 0.01; ***p < 0.001. Fluorescence intensity of TH-immunoreactive fibers was used to estimate striatal TH-fiber density (hatched bars) on the lesioned side, expressed as a percentage of the unlesioned contralateral side. f, As a physiological measure of striatal DA depletion in mock- or 6-OHDA-lesioned animals, rotational behavior induced by an intraperitoneal injection of 2.5 mg/kg amphetamine was measured weekly in all animals and expressed as the number of ipsilateral turns per minute. Values expressed are group mean ± SEM; **p < 0.01.

Figure 2.

Inhibition of soluble TNF signaling in vivo with XENP345 protects DA neurons from LPS-induced death. Low-dose LPS (5 ng/h in normal saline) was infused chronically for 2 weeks into the substantia nigra of CDF/Fischer 344 rats with or without XENP345 (70 ng/h in normal saline). a, Representative sections of microglia activation detected by C3R-positive cells in nigral sections from rat SNpc chronically infused with vehicle, LPS, LPS plus XENP345, or a single bolus of XENP345 (20 μg). Scale bar, 10 μm. A ramified (resting) morphology is evident in vehicle and XENP345 alone brains. LPS/XENP345 coinfused SNpc displayed fewer ameboid (activated) microglia compared with LPS alone. b, Representative sections of TH-IR from an animal infused with LPS and two different animals coinfused with LPS/XENP345. Scale bar, 100 μm. c, Stereological estimates of nigral DA neuron number (TH/NeuN-coimmunoreactive neurons) after LPS or LPS/XENP345 infusion in SNpc on the unlesioned (contralateral) side in solid bars and lesioned (ipsilateral) side shown in gray hatched bars. Left versus right differences from the same animals were analyzed using two-tailed paired Student's t test. Values were expressed as the group mean ± SEM; *p < 0.05, **p < 0.01, significantly different from unlesioned side. d, Striatal TH-IR optical density on infused side (expressed as a percentage of contralateral side). Values expressed are the mean ± SEM; **p < 0.01, significantly different from LPS-only infused group.

Figure 3.

Inhibition of TNF signaling by anti-TNF biologics attenuates LPS-induced neurotoxicity and death of DA neurons in EVM cultures. a, Production of solTNF into culture medium elicited by LPS (10 ng/ml) in rat E14 ventral mesencephalon (EVM) neuron/glia cultures was measured by quantitative ELISA. b, EVM cultures were treated with LPS (10 ng/ml) in medium supplemented with 2.5% FBS (see Materials and Methods) for 4 d in the presence or absence of solTNF-selective XENP345 or etanercept, a soluble decoy human Fc-TNFR2 receptor that inhibits both tmTNF and solTNF. DA neurons were identified as double-labeled TH/MAP2b cells. Two representative panels are shown for vehicle-, LPS-, or LPS- plus XENP345-treated cultures. Similar results were obtained in a minimum of three independent experiments. c, Quantification of DA neuron survival in EVM cultures treated with LPS (10 ng/ml) for 4 d in the presence or absence of TNF inhibitors. Similar results were obtained in a minimum of three independent experiments. Values shown are mean ± SEM; histogram bars with different letters are significantly different (p < 0.05). d, Uptake of [³H]dopamine was measured in LPS-treated EVM cultures in the presence or absence of TNF inhibitors. Values shown are background-corrected for nonspecific uptake measured by including mazindol during incubation with the tritiated neurotransmitter. Values shown are mean + SEM; histogram bars with different letters are significantly different (p < 0.05). Similar results were obtained in three independent experiments. e, GABAergic neuron survival, assessed by counting number of GABA/NeuN double-labeled cells, was unaffected by LPS treatment. No statistical significant difference was found between groups. Similar results were obtained in two independent experiments.

Figure 4.

Inhibition of soluble TNF signaling attenuates 6-OHDA-induced death of DA neurons in EVM cultures. a, Production of solTNF into culture medium elicited by 6-OHDA [0 μm (white), 20 μm (gray), or 50 μm (black)] in rat E14 ventral mesencephalon (EVM) neuron/glia cultures was measured by quantitative ELISA. b, EVM cultures were treated with 6-hydroxydopamine (20 μm) for 24 h in medium supplemented with 2.5% FBS (see Materials and Methods) in the presence or absence of the solTNF-selective XENP345 (200 ng/ml) or etanercept (200 ng/ml). DA neuron survival was assessed 96 h after exposure to 6-OHDA. DA neurons were identified as double-labeled TH/MAP2b cells. Representative panels are shown for vehicle-, 6-OHDA-, 6-OHDA/XENP345-, or 6-OHDA/etanercept-treated cultures. Scale bar, 100 μm. c, Uptake of [³H]dopamine was measured in 6-OHDA-treated EVM cultures in the presence or absence of TNF inhibitors. Values shown are background-corrected for nonspecific uptake measured by including mazindol during incubation with the tritiated neurotransmitter. Values shown are mean ± SEM; histogram bars with different letters are significantly different (p < 0.05).

Figure 5.

Soluble TNF is a primary mediator of LPS-induced microglia activation and death of DA neurons in ventral mesencephalon neuron/glia cultures. a, Quantification of microglia activation (measured by C3R immunoreactivity) induced by LPS treatment for 12, 24, 48, or 96 h in the presence (squares) or absence (circles) of XENP345. Vehicle-treated cultures are indicated by triangles. Values are expressed as the mean ± SEM; error bars are smaller than the symbols; **p < 0.01, ***p < 0.001. Similar results were obtained in at least three independent experiments. b, Representative images of microglia activation measured after 24 h of LPS stimulation in the presence or absence of XENP345 (scale bar, 50 μm) is indicated by number of ameboid-shaped CR3-immunoreactive microglia in the neuron/glia cell cultures. Similar results were obtained in three independent experiments. c, Control experiment showing neutralization of solTNF-induced microglia activation by XENP345 and absence of TNF-like activity when used alone. EVM cultures were treated with TNF (4 ng/ml) in the presence or absence of XENP345. After 24 h stimulation, cells were fixed and stained with an antibody specific for C3R to quantify the extent of microglia activation. Values shown are mean ± SEM; histogram bars with different letters are significantly different (p < 0.05).

Figure 6.

Delayed addition of XENP345 provides robust rescue of DA neurons despite sustained microglia activation. a, Quantification of DA neuron survival after coaddition or delayed addition of XENP345 (200 ng/ml) after LPS (10 ng/ml) treatment. Values shown are mean + SEM; histogram bars with different letters are significantly different (p < 0.05). Similar results were obtained in two independent experiments. b, Quantification of microglia activation in response to LPS (10 ng/ml) in the presence or absence of XENP345 (200 ng/ml) using three different immunocytochemical microglial markers (C3R, CD45, IB4). Similar results were obtained in two independent experiments.