Lauren Costantini

Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation.

In order to assess the potential of embryonic stem cells to undergo neuronal differentiation in vivo, totipotent stem cells from mouse blastocysts (D3 and E14TG2a; previously expanded in the presence of leukemia inhibitory factor) were transplanted, with or without retinoic acid pretreatment, into adult mouse brain, adult lesioned rat brain, and into the mouse kidney capsule. Intracerebral grafts survived in 61% of cyclosporine immunosuppressed rats and 100% of mouse hosts, exhibited variable size and morphology, and both intracerebral and kidney capsule grafts developed large numbers of cells exhibiting neuronal morphology and immunoreactivity for neurofilament, neuron-specific enolase, tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), and cells immunoreactive for glial fibrillary acidic protein. Though graft size and histology were variable, typical grafts of 5-10 mm3 contained 10-20,000 TH+ neurons, whereas dopamine-beta-hydroxylase+ cells were rare. Most grafts also included nonneuronal regions. In intracerebral grafts, large numbers of astrocytes immunoreactive for glial fibrillary acidic protein were present. Both TH+ and 5-HT+ axons from intracerebral grafts grew into regions of the dopamine-lesioned host striatum. TH+ axons grew preferentially into striatal gray matter, while 5-HT+ axons showed no white/gray matter preference. These findings demonstrate that transplantation to the brain or kidney capsule can induce a significant fraction of totipotent embryonic stem cells to become putative dopaminergic or serotonergic neurons and that when transplanted to the brain these neurons are capable of innervating the adult host striatum.

Gene therapy in the CNS

Gene therapy for neurological disorder is currently an experimental concept. The goals for clinical utilization are the relief of symptoms, slowing of disease progression, and correction of genetic abnormalities. Experimental studies are realizing these goals in the development of gene therapies in animal models. Discoveries of the molecular basis of neurological disease and advances in gene transfer systems have allowed focal and global delivery of therapeutic genes for a wide variety of CNS disorders. Limitations are still apparent, such as stability and regulation of transgene expression, and safety of both vector and expressed transgene. In addition, the brain adds several challenges not seen in peripheral gene therapy paradigms, such as post-mitotic cells, heterogeneity of cell types and circuits, and limited access. Moreover, it is likely that several modes of gene delivery will be necessary for successful gene therapies of the CNS. Collaborative efforts between clinicians and basic researchers will likely yield effective gene therapy in the CNS.

Gene Transfer to the Nigrostriatal System by Hybrid Herpes Simplex Virus/Adeno-Associated Virus Amplicon Vectors

To improve gene transfer to CNS neurons, critical elements of herpes simplex virus 1 (HSV-1) amplicons and recombinant adeno-associated virus (AAV) vectors were combined to construct a hybrid amplicon vector, and then packaged via a helper virus-free system. We tested the HSV/AAV hybrid amplicon vectors for transduction efficiency and stability of transgene expression (green fluorescent protein) in primary neuronal cultures from rat fetal ventral mesencephalon, in comparison with traditional HSV amplicon, AAV, or adenovirus (Ad) vectors at the same multiplicity of infection. The HSA/AAV hybrid vectors transduced the highest number of primary neurons in culture 2 days after infection. As compared with all other vectors tested, only hybrid vectors containing the AAV rep gene maintained the 2-day level of transgene expression over 12 days in culture. This rep-containing hybrid vector was then tested for efficiency and safety in the brain. One month after injection into adult rat striatum (1 x 10(6) transducing units injected), transgene expression was observed within the striatum (ranging from 564 to 8610 cells) and the substantia nigra (via retrograde transport, ranging from 130 to 809 neurons). The HSV/AAV hybrid amplicon vectors transduced predominantly neurons within the striatum, and showed transduction efficacy similar to and in many cases higher than that of HSV amplicon vectors. No immune response was observed in the HSA/AAV hybrid vector-injected brains, as determined by immune markers specific for helper T lymphocytes, cytotoxic T lymphocytes, and microglia. This HSV/AAV hybrid system shows high transduction efficiency and stability in culture. The effective and safe transgene delivery into the nigrostriatal system illustrates its potential for therapeutic application for neurologic disorders, such as Parkinson and Huntington disease.

Immunophilin ligands and GDNF enhance neurite branching or elongation from developing dopamine neurons in culture.

Neurotrophic effects of immunophilin ligands have been shown in animal models of peripheral and central nervous system insult. To investigate the specific growth-promoting effects of these compounds, we examined the effects of various immunophilin ligands on primary dopamine (DA) neurons in culture and compared these with a well-known DA trophic factor, glial cell line-derived neurotrophic factor (GDNF). In neuronal cultures from Embryonic Day 14 ventral mesencephalon, enhanced elongation of DA neurites was observed with immunophilin ligands, which inhibited the phosphatase activity of calcineurin (FK506 and cyclosporin A) when compared to vehicle-treated cultures. This elongation was also observed with GDNF, known to exert its trophic effects through phosphorylation-dependent pathways. In contrast, immunophilin ligands that do not inhibit calcineurin (rapamycin and V-10,367) increased branching of DA neurites, suggesting that elongation is dependent upon maintained phosphorylation while branching is not. In addition, both V-10,367 and rapamycin antagonized the elongation effects of FK506 and induced branching. The antagonism of elongation (and reappearance of branching) illustrates the intrinsic abilities of developing DA neurons to either elongate or branch, but not both. We show that the immunophilin FKBP12 (12-kDa FK506-binding protein) is expressed in ventral mesencephalic neuronal cultures and colocalizes with DA neurons. This work elucidates the specific growth-promoting effects by which GDNF and immunophilin ligands modify developmental growth processes of DA neurons, via their interactions with intracellular targets.

Immunophilin ligands can prevent progressive dopaminergic degeneration in animal models of Parkinson's disease.

Slowing or halting the progressive dopaminergic (DA) degeneration in Parkinsons disease (PD) would delay the onset and development of motor symptoms, prolong the efficacy of pharmacotherapies and decrease drug‐induced side‐effects. We tested the potential of two orally administered novel immunophilin ligands to protect against DA degeneration in two animal models of PD. First, in an MPTP (N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) mouse model, we compared an immunophilin ligand (V‐10,367) documented to bind the immunophilin FKBP12 with V‐13,661, which does not bind FKBP12. Both molecules could prevent the loss of striatal DA innervation in a dose‐dependent fashion during 10 days of oral administration. Second, to determine whether an immunophilin ligand can protect against progressive and slow DA degeneration typical of PD, an intrastriatal 6‐hydroxydopamine‐infusion rat model was utilized. Oral treatment with the FKBP12‐binding immunophilin ligand began on the day of lesion and continued for 21 days. At this time point, post mortem analyses revealed that the treatment had prevented the progressive loss of DA innervation within the striatum and loss of DA neurons within the substantia nigra, related to functional outcome as measured by rotational behaviour. Notably, DA fibres extending into the area of striatal DA denervation were observed only in rats treated with the immunophilin ligand, indicating neuroprotection or sprouting of spared DA fibres. This is the first demonstration that immunophilin ligands can prevent a slow and progressive DA axonal degeneration and neuronal death in vivo. The effects of orally administered structurally related immunophilin ligands in acute and progressive models of DA degeneration are consistent with the idea that these compounds may have therapeutic value in PD.

Immune parameters relevant to neural xenograft survival in the primate brain

Cicchetti F, Fodor W, Deacon TW, van Horne C, Rollins S, Burton W, Costantini LC, Isacson O. Immune parameters relevant to neural xenograft survival in the primate brain. Xenotransplantation 2003: 10: 41–49.

Cell implantation therapies for Parkinson's disease using neural stem, transgenic or xenogeneic donor cells

A new therapeutic neurological and neurosurgical methodology involves cell implantation into the living brain in order to replace intrinsic neuronal systems, that do not spontaneously regenerate after injury, such as the dopaminergic (DA) system affected in Parkinsons disease (PD) and aging. Current clinical data indicate proof of principle for this cell implantation therapy for PD. Furthermore, the disease process does not appear to negatively affect the transplanted cells, although the patients endogenous DA system degeneration continues. However, the optimal cells for replacement, such as highly specialized human fetal dopaminergic cells capable of repairing an entire degenerated nigro-striatal system, cannot be reliably obtained or generated in sufficient numbers for a standardized medically effective intervention. Xenogeneic and transgenic cell sources of analogous DA cells have shown great utility in animal models and some promise in early pilot studies in PD patients. The cell implantation treatment discipline, using cell fate committed fetal allo- or xenogeneic dopamine neurons and glia, is currently complemented by research on potential stem cell derived DA neurons. Understanding the cell biological principles and developing methodology necessary to generate functional DA progenitors is currently our focus for obtaining DA cells in sufficient quantities for the unmet cell transplantation need for patients with PD and related disorders.

Combined Inhibition of Apoptosis and Complement Improves Neural Graft Survival of Embryonic Rat and Porcine Mesencephalon in the Rat Brain

To define potential mechanisms of cell death during neural cell transplantation, we investigated the role of intracellular caspase activation in combination with the activation of serum complement. We demonstrated that ventral mesencephalic (VM) cells are susceptible to complement-mediated cell lysis that can be blocked with an anti-C5 complement inhibitor (18A10). We also determined that incubating freshly isolated allogenic VM cells with the caspase inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), followed by immediate striatal implantation, led to a 2.5-fold increase in tyrosine hydroxylase (TH) cell survival 12 weeks postimplantation (P < 0.05). In contrast, overnight incubation with BAF followed by striatal implantation led to a 2-fold reduction in TH cell survival at 12 weeks (P < 0.05). Using the optimal BAF treatment and complement inhibition, we tested the hypothesis that these treatments would lead to increased cell survival in both allogeneic and xenogeneic transplantation models. We transplanted cell suspensions of (a) rat E14 VM or VM treated with (b) BAF alone, (c) anti-C5, or (d) a combination of BAF and anti-C5. There was a significant increase in the relative number of TH-positive cells in the BAF/anti-C5 group versus control at 12 weeks posttransplantation. Similar results were achieved in a pig to rat xenotransplant paradigm. A neuronal xenograft marker (70-kDa neurofilament) also demonstrated relative increases in graft volume in the BAF/anti-C5 treatment group. These studies indicate that more than one mechanism can mediate cell death during neural cell transplantation and that a combined treatment using caspase and complement inhibition can significantly improve cell survival.

Enhanced axonal growth from fetal human bcl-2 transgenic mouse dopamine neurons transplanted to the adult rat striatum

Embryonic neurons transplanted to the adult CNS extend axons only for a developmentally defined period. There are certain intercellular factors that control the axonal extension, one of which may be the expression of the bcl-2 protein. In this study, rats with complete striatal dopamine fiber denervation received embryonic day 14 mouse ventral mesencephalon cells overexpressing human bcl-2 or control wild-type ventral mesencephalon cells. All rats were treated with cyclosporine to prevent rejection and the surviving grafts were analyzed for cell survival and outgrowth of dopaminergic fibers. The results demonstrate that bcl-2 overexpression does not enhance neuronal graft survival. However, the bcl-2 overexpressing neurons had a higher number of dopaminergic fibers that grew longer distances. These results show that overexpression of bcl-2 can result in longer distance axonal growth of transplanted fetal dopaminergic neurons and that genetic modification of embryonic donor cells may enhance their ability to reinnervate a neuronal target territory.

Medial fetal ventral mesencephalon: a preferred source for dopamine neuron grafts.

CURRENTLY, fetal tissue transplantation into patients with Parkinsons disease utilizes the entire ventral mesencephalon (VM) as donor tissue. However, the resulting mixture of cell types contains a relatively low proportion of therapeutically relevant dopamine (DA) neurons. We show that differential dissection of a medial region of embryonic day 14 rat VM yields a significantly higher proportion of DA neurons (8–10%) than is found in lateral VM (2%) or whole VM (4–5%). Medial VM also contained a larger number of the specific subpopulation of DA neurons (aldehyde dehydrogenase-positive; AHD) that project to dorsolateral motor region of the striatum. Selective dissection of fetal medial VM selectively enriches DA neurons in cell preparations useful for transplantation in Parkinsons disease.