Susan O. McGuire

Tumor Necrosis Factor α Is Toxic to Embryonic Mesencephalic Dopamine Neurons

Abstract Levels of the proinflammatory cytokine tumor necrosis factor α (TNFα) are increased in postmortem brain and cerebral spinal fluid from patients with Parkinsons disease (PD). This observation provides a basis for associating TNFα with neurodegeneration, but a specific toxicity in dopamine (DA) neurons has not been firmly established. Therefore, we investigated TNFα-induced toxicity in DA neurons by utilizing primary cultures of embryonic rat mesencephalon. Exposure to TNFα resulted in a dose-dependent decrease in DA neurons as evidenced by decreased numbers of tyrosine hydroxylase-immunoreactive (THir) cells. TNFα toxicity was selective for DA neurons in that neither glial cell counts nor the total number of neurons was decreased and no general cytotoxicity was evidenced by lactate dehydrogenase assay. Many of the cells which remained immunoreactive for TH had shrunken and rounded cell bodies with broken, blunted, or absent processes. However, TNFα-treated cultures also contained some THir cells which appeared to be undamaged and possibly resistant to TNFα-induced toxicity. Additionally, immunocytochemistry revealed basal expression of TNFα receptor 1 (p55, R1) and TNFα receptor 2 (p75, R2) on all cells within the mesencephalic cultures to some degree, even though only DA neurons were affected by TNFα treatment. These data strongly suggest that TNFα mediates cell death in a sensitive population of DA neurons and support the potential involvement of proinflammatory cytokines in the degeneration of DA neurons in PD.

A clonal line of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines: a source of cells for transplantation in Parkinson's disease.

Neural progenitor cells potentially provide a limitless, on-demand source of cells for grafting into patients with Parkinsons disease (PD) if the signals needed to control their conversion into dopamine (DA) neurons could be identified. We have recently shown that cytokines which instruct cell division and differentiation within the hematopoeitic system may provide similar functions in the central nervous system. We have shown that mitotic progenitor cells can be isolated from embryonic rat mesencephalon and that these cells respond to a combination of interleukin-1, interleukin-11, leukemia inhibitory factor, and glial cell line-derived neurotrophic factor yielding a tyrosine hydroxylase-immunoreactive (THir) phenotype in 20-25% of total cells. In the present study, 24 clonal cell lines derived from single cells of mesencephalic proliferation spheres were examined for their response to the cytokine mixture. The clone yielding the highest percentage of THir neurons (98%) was selected for further study. This clone expressed several phenotypic characteristics of DA neurons and expression of Nurr1. The response to cytokines was stable for several passages and after cryopreservation for several months. When grafted into the striatum of DA-depleted rats, these cells attenuated rotational asymmetry to the same extent as freshly harvested embryonic DA neurons. These data demonstrate that mesencephalic progenitor cells can be clonally expanded in culture and differentiated in the presence of hematopoietic cytokines to yield enriched populations of DA neurons. When transplanted, these cells provide significant functional benefit in the rat model of PD.

Neuroprotective effects of D3 dopamine receptor agonists

The effects of D(3) receptor activation are unresolved at this time, but may have practical implications in the treatment of Parkinsons disease (PD). As a result of assessing the neuroprotective effects of the direct-acting D(3) preferring dopamine (DA) agonist pramipexole (PPX), we have observed that drugs which psossess D(3) affinity increase the production of a DA neurotrophic factor in tissue culture. This molecule is increased by treatment with PPX, is constitutively produced by DA neurons in culture, and possesses a molecular weight of approximately 35kDa. It is hypothesized that this molecule may be the so-called DA autotrophic factor referred to by many authors over the past two decades. Interestingly, the protein is oxidant-labile and, therefore, D(3) agonists which increase its production and also possess antioxidant capacity would provide unique neuroprotective benefits to patients with PD. However, many questions remain. Although the data supporting this notion are strong, it is clear that other unknown characteristics of DA agonists, including increased production of anti-apoptotic proteins, are also involved. This manuscript will review this concept in the context of tissue culture strategies of neuroprotection. Although no conclusion can be made at this time, it is clear that direct comparisons of the neuroprotective effects of direct-acting DA agonists in mesencephalic culture can provide considerable insight into the mechanistic actions of anti-dopaminergic drugs.

Angiogenic and neurotrophic effects of vascular endothelial growth factor (VEGF165): studies of grafted and cultured embryonic ventral mesencephalic cells

The present series of experiments investigated the effects of vascular endothelial growth factor (VEGF165) on adult rat striatal cerebrovasculature and embryonic dopamine (DA) neuron allografts in a rat model of Parkinsons disease (PD). We examined VEGF165s ability to (1) alter the vascular network of the adult rat striatum, (2) influence the vascular growth of solid embryonic day 14 (E14) ventral mesencephalic (VM) grafts when placed into a VEGF-pretreated host striatum, (3) alter the function and survival of E14 VM grafts when transplanted into an adult DA-deleted striatum, and (4) influence cell survival and neurite growth in cultures of E14 VM cells. We demonstrate here that a single bolus injection of VEGF165 into the adult rat striatum significantly increases the amount of vasculature in the vicinity of the injection site in a delayed and transient manner when compared to saline controls. Transplanting solid E14 VM grafts into the VEGF165-pretreated striatum resulted in a homogeneous distribution of small blood vessels throughout the graft, a pattern that closely resembles mature adult vasculature. In contrast, grafts in the control condition contained a patchy distribution of heavily dilated vessels. Behavioral measurements indicate that VEGF pretreatment of the intrastriatal graft site accelerates recovery of amphetamine-induced rotational asymmetry in unilateral 6-OHDA lesioned rats. Unexpectedly, however, VEGF pretreatments failed to increase survival of tyrosine hydroxylase-immunoreactive (THir) neurons in the grafts. In contrast to this finding in vivo, adding VEGF165 to glial-reduced E14 rat VM cultures produced a fourfold increase in THir cell survival and a doubling in the length of THir neurites. We conclude that with the proper method of delivery, VEGF165 may prove to be one of several strategies necessary to significantly improve the survival and function of fetal VM tissue grafts.

Oligodendrocyte-type 2 astrocyte-derived trophic factors increase survival of developing dopamine neurons through the inhibition of apoptotic cell death

Survival of embryonic dopamine (DA) neurons is extremely low (5–20%) following transplantation. Strategies to increase this survival are critical to the future of transplantation for Parkinsons disease. We demonstrate here that a factor(s) released from striatal oligodendrocyte‐type 2 astrocytes (SO2A) greatly improves the survival and phenotype expression of mesencephalic DA neurons in culture while simultaneously decreasing the presence of apoptotic nuclear profiles, as detected by the TUNEL method and bisbenzamide/tyrosine hydroxylase double labeling. This SO2A‐derived trophic factor(s) has minimal effects on glia and no effect on nondopaminergic mesencephalic neurons. The developmental period during which this SO2A trophic effect occurs (E14–18) coincides with the period when mesencephalic grafts are undergoing the highest rates of apoptosis, i.e., immediately following implantation. Therefore, SO2A‐derived trophic factor(s) offers great potential for the augmentation of grafted DA neuron survival. J. Comp. Neurol. 426:143–153, 2000.

The synaptic impact of the host immune response in a parkinsonian allograft rat model : Influence on graft-derived aberrant behaviors

Graft-induced dyskinesias (GIDs), side-effects found in clinical grafting trials for Parkinsons disease (PD), may be associated with the withdrawal of immunosuppression. The goal of this study was to determine the role of the immune response in GIDs. We examined levodopa-induced dyskinesias (LIDs), GID-like behaviors, and synaptic ultrastructure in levodopa-treated, grafted, parkinsonian rats with mild (sham), moderate (allografts) or high (allografts plus peripheral spleen cell injections) immune activation. Grafts attenuated amphetamine-induced rotations and LIDs, but two abnormal motor syndromes (tapping stereotypy, litter retrieval/chewing) emerged and increased with escalating immune activation. Immunohistochemical analyses confirmed immune activation and graft survival. Ultrastructural analyses showed increases in tyrosine hydroxylase-positive (TH+) axo-dendritic synapses, TH+ asymmetric specializations, and non-TH+ perforated synapses in grafted, compared to intact, striata. These features were exacerbated in rats with the highest immune activation and correlated statistically with GID-like behaviors, suggesting that immune-mediated aberrant synaptology may contribute to graft-induced aberrant behaviors.

Interference with anoikis‐induced cell death of dopamine neurons: Implications for augmenting embryonic graft survival in a rat model of Parkinson's disease

One promising therapy for the treatment of Parkinsons disease is transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted cells die, many via apoptosis. In this study we attempted to prevent anoikis‐induced cell death, which is triggered during the preparation of cells for grafting, and examine the impact on graft viability and function. We utilized the extracellular matrix molecule tenascin‐C (tenascin) and an antibody (Ab) to the cell adhesion molecule L1 to specifically mimic survival signals induced by cell–matrix and cell–cell interactions. In vitro, both tenascin‐ and L1 Ab‐treated cultures doubled the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to control. Additionally, cell survival assays determined that tenascin and L1 Ab‐treated cell suspensions yielded more metabolically active and fewer dead cells than control suspensions. In contrast to the culture results, tenascin‐ and L1 Ab‐treated mesencephalic grafts did not yield an increase in the number of THir neurons using our standard grafting paradigm (3 μl of 100,000 cells/μl). However, under low‐density conditions (3 μl of 3,000 cells/μl), tenascin augmented grafted THir neuron survival. These findings are consistent with the view that cell density can dramatically influence the degree of stress placed on THir neurons and consequently affect the success of survival strategies in vivo. In conclusion, pretreatment with tenascin may prove beneficial to prevent anoikis in dilute cell suspension grafts, while long‐term in vivo delivery methods need to be explored to determine if L1 can prevent anoikis in grafts of mesencephalic dopamine neurons after transplantation. J. Comp. Neurol. 464:172–179, 2003.

Exogenous erythropoietin provides neuroprotection of grafted dopamine neurons in a rodent model of Parkinson's disease

Parkinsons disease (PD) is a neurodegenerative disease marked by severe loss of dopamine (DA) neurons in the nigrostriatal system, which results in depletion of striatal DA. Transplantation of embryonic ventral mesencephalic (VM) DA neurons into the striatum is a currently explored experimental treatment aimed at replacing lost DA in the nigrostriatal system, but is plagued with poor survival (5-20%) of implanted neurons. Here, we tested the ability of erythropoietin (Epo) to provide neuroprotection for embryonic day 14 (E14) VM DA neurons. Epo was tested in vitro for the ability to augment tyrosine hydroxylase-immunoreactive (TH-ir) neuron survival under normal cell culture conditions. In vitro, Epo did not increase the number of TH-ir neurons when administered at the time of plating the E14 VM cells in culture. We also tested the efficacy of Epo to enhance E14 VM transplants in vivo. Rats unilaterally lesioned with 6-hydroxydopamine received transplants that were incubated in Epo. Treatment with Epo produced significant increases in TH-ir neuron number, soma size, and staining intensity. Animals receiving Epo-treated grafts exhibited significantly accelerated functional improvements and significantly greater overall improvements from rotational asymmetry compared to control grafted rats. These data indicate that the survival of embryonic mesencephalic TH-ir neurons is increased when Epo is administered with grafted cells in a rodent model of PD. As direct neurotrophic effects of Epo were not observed in vitro, the mechanism of Epo neuroprotection remains to be elucidated.

Cellular models to study dopaminergic injury responses

Abstract: The study of immature midbrain dopamine (DA) neurons and dopaminergic cell lines in culture provides an opportunity to analyze mechanisms of cell death and avenues of potential intervention relevant to Parkinsons disease (PD) in a controlled environment. Use of cell culture models has provided evidence for different sets of intracellular changes associated with DA neuron death following exposure to the neurotoxins 6‐hydroxydopamine and MPP+, supporting roles for oxidative stress and impaired energy metabolism as significant factors endangering these cells. Interference with death of cultured DA neurons has provided an initial test system that has yielded all the identified neurotrophic factors for DA neurons. More recent work suggests that combinations of molecules secreted by myelinating glial cells and their precursors provide even greater neuroprotection for DA neurons. Most recently, culture systems have been used to implicate microglial activation in DA neuron injury, providing impetus to the investigation of antiinflammatory agents as potential therapeutics for PD. Thus, cell culture models provide an important bidirectional link between mechanistic studies and clinically relevant observations.

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons.

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH‐immunoreactive (ir) neurons in VM cultures. However, 1 µm GAL reduced an approximately 100% increase in TH‐ir neurons in 1 mm dibutyryl cAMP (dbcAMP)‐treated cultures by ∼50%. TH‐ir neuron number in dbcAMP‐treated VM cultures was dose‐responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi‐quantitative RT‐PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific ∼200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)‐sensitive opening of G protein‐gated K+ channels (GIRKs). GAL reduction of TH‐ir neuron number in dbcAMP + GAL‐treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1‐ mediated reduction of TH in midbrain dopaminergic neurons.