The expression of soluble growth and survival promoting factors by neural precursor cells (NPCs) is suggested to be a prominent mechanism underlying the protective and regenerative effects of these cells after transplantation. Nevertheless, how and to what extent specific NPC-expressed factors contribute to therapeutic effects is not well understood. Using RNA silencing, the current study investigated the roles of two donor NPC molecules, namely glial cell-line derived neurotrophic factor (GDNF) and sonic hedgehog (SHH), in the protection of substantia nigra dopamine neurons in rats treated with 6-hydroxydopamine (6-OHDA). Analyses indicate that as opposed to the knock-down of GDNF, SHH inhibition caused a profound decline in nigrostriatal neuroprotection. Further, SHH silencing also curbed endogenous neurogenesis and the migration of host brdU+/dcx+ neural precursors into the striatum, which was present in the animals receiving control or GDNF silenced NPCs. A change in graft phenotype, mainly reflected by a reduced proportion of undifferentiated nestin+ cells, as well as a significantly greater host microglial activity, suggested an important role for these processes in the attenuation of neuroprotection and neurogenesis upon SHH silencing. Overall these studies reveal core mechanisms fundamental to grafted NPC-based therapeutic effects, and delineate the particular contributions of two graft-expressed molecules, SHH and GDNF, in mediating midbrain dopamine neuron protection, and host plasticity after NPC transplantation.
The discovery of biomarkers for Parkinson’s disease (PD) is challenging due to the heterogeneous nature of this disorder, and a poor correlation between the underlying pathology and the clinically expressed phenotype. An ideal biomarker would inform on PD-relevant pathological changes via an easily assayed biological characteristic, which reliably tracks clinical symptoms. Human dermal (skin) fibroblasts are accessible peripheral cells that constitute a patient-specific system, which potentially recapitulates the PD chronological and epigenetic aging history. Here, we compared primary skin fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls. These fibroblasts were studied from fundamental viewpoints of growth and morphology, as well as redox, mitochondrial, and autophagic function. It was observed that fibroblasts from PD subjects had higher growth rates, and appeared distinctly different in terms of morphology and spatial organization in culture, compared to control cells. It was also found that the PD fibroblasts exhibited significantly compromised mitochondrial structure and function when assessed via morphological and oxidative phosphorylation assays. Additionally, a striking increase in baseline macroautophagy levels was seen in cells from PD subjects. Exposure of the skin fibroblasts to physiologically relevant stress, specifically ultraviolet irradiation (UVA), further exaggerated the autophagic dysfunction in the PD cells. Moreover, the PD fibroblasts accumulated higher levels of reactive oxygen species (ROS) coupled with lower cell viability upon UVA treatment. In essence, these studies highlight primary skin fibroblasts as a patient-relevant model that captures fundamental PD molecular mechanisms, and supports their potential utility to develop diagnostic and prognostic biomarkers for the disease.
Neuroprotective and neurorescue effects after neural stem/precursor cell (NPC) transplantation have been reported, but the mechanisms underlying such phenomena are not well understood. Our recent findings in a rat Parkinson's disease (PD) model indicate that transplantation of NPCs before a 6-hydroxydopamine (6-OHDA) insult can result in nigrostriatal protection which is associated with endogenous NPC proliferation, migration and neurogenesis. Here, we sought to determine whether the observed endogenous NPC response (i) contributes to transplanted NPC-mediated neuroprotection; and/or (ii) affects graft phenotype and function. Host Fischer 344 rats were administered the antimitotic agent cytosine-β-d-arabinofuranoside (Ara-C) to eliminate actively proliferating endogenous neural precursors before being transplanted with NPCs and treated with 6-OHDA to induce nigrostriatal degeneration. Behavioral and histological analyses demonstrate that the neuroprotective response observed in NPC transplanted animals which had not received Ara-C was significantly attenuated in animals which did receive pre-transplant Ara-C. Also, while grafts in Ara-C-treated animals showed no decrease in cell number, they exhibited significantly reduced expression of the neural stem cell regulators nestin and sonic hedgehog. In addition, inhibition of the endogenous NPC response resulted in an exaggerated host glial reaction. Overall, the study establishes for the first time that endogenous NPCs contribute to transplanted NPC-mediated therapeutic effects by affecting both grafted and mature host cells in unique ways. Thus, both endogenous and transplanted NPCs are important in creating an environment suitable for neural protection and rescue, and harnessing their synergistic interaction may lead to the optimization of cell-based therapies for PD.
Realistically, future stem cell therapies for neurological conditions including Parkinson's disease (PD) will most probably entail combination treatment strategies, involving both the stimulation of endogenous cells and transplantation. Therefore, this study investigates these two modes of neural precursor cell (NPC) therapy in concert in order to determine their interrelationships in a rat PD model. Human placental alkaline phosphatase (hPAP)-labeled NPCs were transplanted unilaterally into host rats which were subsequently infused ipsilaterally with 6-hydroxydopamine (6-OHDA). The reaction of host NPCs to the transplantation and 6-OHDA was tracked by bromodeoxyuridine (BrdU) labeling. Two weeks after transplantation, in animals transplanted with NPCs we found evidence of elevated host subventricular zone NPC proliferation, neurogenesis, and migration to the graft site. In these animals, we also observed a significant preservation of striatal tyrosine hydroxylase (TH) expression and substantia nigra TH cell number. We have seen no evidence that neuroprotection is a product of dopamine neuron replacement by NPC-derived cells. Rather, the NPCs expressed glial cell line-derived neurotrophic factor (GDNF), sonic hedgehog (Shh), and stromal cell-derived factor 1 alpha (SDF1alpha), providing a molecular basis for the observed neuroprotection and endogenous NPC response to transplantation. In summary, our data suggests plausible synergy between exogenous and endogenous NPC actions, and that NPC implantation before the 6-OHDA insult can create a host microenvironment conducive to stimulation of endogenous NPCs and protection of mature nigral neurons.
Here we investigated the neuroprotective potential of systemic CD34(+) human cord blood cells (hCBCs) in a 6-hydroxydopamine rat model of Parkinson's disease.