Blocking Soluble Tumor Necrosis Factor Signaling with Dominant-Negative Tumor Necrosis Factor Inhibitor Attenuates Loss of Dopaminergic Neurons in Models of Parkinson's Disease
Melissa K. McCoy, Terina N. Martinez, Kelly A. Ruhn, David E. Szymkowski, Christine G. Smith et al.
Objective: To evaluate the role of soluble TNF signaling in retrograde nigral degeneration induced by striatal 6-OHDA injection, and to assess neuroprotective effects of TNF inhibition in a Parkinson's disease model
Materials & Equipment Checklist
7 items1 from ConductScience
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Equipment2
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View 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.
Protocol Steps
1
Striatal 6-OHDA injection
Inject 6-hydroxydopamine into the striatum to induce retrograde nigral degeneration
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Note: This is the primary neurotoxic insult to model Parkinson's disease
View evidence from paper
“retrograde nigral degeneration induced by striatal injection of the oxidative neurotoxin 6-hydroxydopamine (6-OHDA)”
2
Nigral XENP345 infusion
Infuse XENP345 into the substantia nigra to neutralize soluble TNF
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Note: XENP345 was neuroprotective only when infused into the nigra, not the striatum
View evidence from paper
“XENP345 was neuroprotective only when infused into the nigra, not the striatum”
Chronically coinfuse XENP345 with bacterial lipopolysaccharide into the substantia nigra
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Note: Used to confirm role of solTNF-dependent neuroinflammation in nigral degeneration
View evidence from paper
“Similar protective effects were observed with chronic in vivo coinfusion of XENP345 with bacterial lipopolysaccharide (LPS) into the substantia nigra”
5
In vitro embryonic midbrain culture preparation
Prepare embryonic rat midbrain neuron/glia cell cultures for in vitro studies
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Note: Cultures exposed to LPS to model neuroinflammation
View evidence from paper
“In embryonic rat midbrain neuron/glia cell cultures exposed to LPS, even delayed administration of XENP345 prevented selective degeneration of DA neurons”
6
Delayed XENP345 administration in vitro
Administer XENP345 with delayed timing in LPS-exposed midbrain cultures
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Note: Demonstrates neuroprotection even with delayed treatment despite sustained microglia activation
View evidence from paper
“even delayed administration of XENP345 prevented selective degeneration of DA neurons despite sustained microglia activation and secretion of solTNF”
7
In vitro 6-OHDA toxicity assessment
Expose midbrain cultures to 6-OHDA and assess XENP345 neuroprotective effects
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Note: Evaluates TNF role in 6-OHDA-induced dopaminergic neuron toxicity
View evidence from paper
“XENP345 also attenuated 6-OHDA-induced DA neuron toxicity in vitro”
Subjects / Specimens
Species
rat
Strain
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Age
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Sex
unknown
Weight
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Embryonic rat midbrain neuron/glia cell cultures also used for in vitro studies