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 Parkinson's 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 patient's 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.

Porcine xenografts in Parkinson's disease and Huntington's disease patients: preliminary results.

The observation that fetal neurons are able to survive and function when transplanted into the adult brain fostered the development of cellular therapy as a promising approach to achieve neuronal replacement for treatment of diseases of the adult central nervous system. This approach has been demonstrated to be efficacious in patients with Parkinson's disease after transplantation of human fetal neurons. The use of human fetal tissue is limited by ethical, infectious, regulatory, and practical concerns. Other mammalian fetal neural tissue could serve as an alternative cell source. Pigs are a reasonable source of fetal neuronal tissue because of their brain size, large litters, and the extensive experience in rearing them in captivity under controlled conditions. In Phase I studies porcine fetal neural cells grafted unilaterally into Parkinson's disease (PD) and Huntington's disease (HD) patients are being evaluated for safety and efficacy. Clinical improvement of 19% has been observed in the Unified Parkinson's Disease Rating Scale "off" state scores in 10 PD patients assessed 12 months after unilateral striatal transplantation of 12 million fetal porcine ventral mesencephalic (VM) cells. Several patients have improved more than 30%. In a single autopsied PD patient some porcine fetal VM cells were observed to survive 7 months after transplantation. Twelve HD patients have shown a favorable safety profile and no change in total functional capacity score 1 year after unilateral striatal placement of up to 24 million fetal porcine striatal cells. Xenotransplantation of fetal porcine neurons is a promising approach to delivery of healthy neurons to the CNS. The major challenges to the successful use of xenogeneic fetal neuronal cells in neurodegenerative diseases appear to be minimizing immune-mediated rejection, management of the risk of xenotic (cross-species) infections, and the accurate assessment of clinical outcome of diseases that are slowly progressive.

Transplantation of embryonic porcine mesencephalic tissue in patients with PD.

OBJECTIVE: To assess the safety and the effect on standardized clinical rating measures of transplanted embryonic porcine ventral mesencephalic (VM) tissue in advanced PD. METHODS: Twelve patients with idiopathic PD underwent unilateral implantation of embryonic porcine VM tissue; six received cyclosporine immunosuppression and six received tissue treated with a monoclonal antibody directed against major histocompatibility complex class I. Patients were followed for 12 months and assessed by clinical examination, MRI, and 18F-levodopa PET. Porcine endogenous retrovirus testing was conducted by PCR-based method on peripheral blood mononuclear cells. RESULTS: Cell implantation occurred without serious adverse events in all patients. Cultures were negative for bacterial and unknown viral contamination. No porcine endogenous retrovirus DNA sequences were found. MRI demonstrated cannula tracts within the putamen and caudate, with minimal or no edema and no mass effect at the transplant sites. In the medication-off state, total Unified Parkinson's Disease Rating Scale scores improved 19% (p = 0.01). Three patients improved over 30%. There were two patients with improved gait. 18F-levodopa PET failed to show changes on the transplanted side. CONCLUSIONS: Unilateral transplantation of porcine embryonic VM cells into PD patients was well tolerated with no evidence of transmission of porcine endogenous retrovirus. Changes in standardized clinical PD rating measures were variable, similar to the results of the first trials of unilateral human embryonic allografts that transplanted small amounts of tissue.

Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans.

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants "mat" for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.

Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein.

In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).

AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos.

An emerging family of kinases related to the Drosophila Aurora and budding yeast Ipl1 proteins has been implicated in chromosome segregation and mitotic spindle formation in a number of organisms. Unlike other Aurora/Ipl1-related kinases, the Caenorhabditis elegans orthologue, AIR-2, is associated with meiotic and mitotic chromosomes. AIR-2 is initially localized to the chromosomes of the most mature prophase I-arrested oocyte residing next to the spermatheca. This localization is dependent on the presence of sperm in the spermatheca. After fertilization, AIR-2 remains associated with chromosomes during each meiotic division. However, during both meiotic anaphases, AIR-2 is present between the separating chromosomes. AIR-2 also remains associated with both extruded polar bodies. In the embryo, AIR-2 is found on metaphase chromosomes, moves to midbody microtubules at anaphase, and then persists at the cytokinesis remnant. Disruption of AIR-2 expression by RNA- mediated interference produces entire broods of one-cell embryos that have executed multiple cell cycles in the complete absence of cytokinesis. The embryos accumulate large amounts of DNA and microtubule asters. Polar bodies are not extruded, but remain in the embryo where they continue to replicate. The cytokinesis defect appears to be late in the cell cycle because transient cleavage furrows initiate at the proper location, but regress before the division is complete. Additionally, staining with a marker of midbody microtubules revealed that at least some of the components of the midbody are not well localized in the absence of AIR-2 activity. Our results suggest that during each meiotic and mitotic division, AIR-2 may coordinate the congression of metaphase chromosomes with the subsequent events of polar body extrusion and cytokinesis.

A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos.

S. cerevisiae Ipl1, Drosophila Aurora, and the mammalian centrosomal protein IAK-1 define a new subfamily of serine/threonine kinases that regulate chromosome segregation and mitotic spindle dynamics. Mutations in ipl1 and aurora result in the generation of severely aneuploid cells and, in the case of aurora, monopolar spindles arising from a failure in centrosome separation. Here we show that a related, essential protein from C. elegans, AIR-1 (Aurora/Ipl1 related), is localized to mitotic centrosomes. Disruption of AIR-1 protein expression in C. elegans embryos results in severe aneuploidy and embryonic lethality. Unlike aurora mutants, this aneuploidy does not arise from a failure in centrosome separation. Bipolar spindles are formed in the absence of AIR-1, but they appear to be disorganized and are nucleated by abnormal-looking centrosomes. In addition to its requirement during mitosis, AIR-1 may regulate microtubule-based developmental processes as well. Our data suggests AIR-1 plays a role in P-granule segregation and the association of the germline factor PIE-1 with centrosomes.

Spermatid perinuclear ribonucleic acid-binding protein binds microtubules in vitro and associates with abnormal manchettes in vivo in mice.

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The RNA target of SPNR in vivo is unknown, although we have previously suggested the possibility that SPNR is involved in the translational activation of the protamine 1 mRNA in elongated spermatids. To increase our understanding of SPNR's association with the manchette, we sought to determine SPNR's subcellular localization in several mouse mutants that show reduced fertility or sterility and that have structurally abnormal manchettes. We show here that despite the highly abnormal manchettes and microtubule aggregates formed in azh, hop-sterile, tw2, and tw8 mutants, SPNR remains associated with the manchettes. Localization of SPNR to the abnormal manchettes suggests that SPNR is tightly bound to the manchette. SPNR could bind manchette microtubules directly, or it could bind indirectly via an interaction with a microtubule-associated protein (MAP). We sought to determine whether SPNR binds microtubules in vitro, and if so, whether it requires a MAP. We show by Western analysis that the endogenous SPNR protein can be pelleted with murine testis microtubules in a taxol-dependent manner in vitro. A recombinant version of SPNR produced in bacteria can also be pelleted with testis microtubules, as well as microtubules polymerized from purified bovine brain tubulin, an association that is salt-sensitive. These results suggest that SPNR, in addition to its function as an RNA-binding protein, is also a bona fide MAP.

Distribution of Tenr, an RNA-binding protein, in a lattice-like network within the spermatid nucleus in the mouse.

In a molecular screen for cDNAs that encode protamine RNA-binding proteins, we obtained seven independent clones that encode Tenr, a testis nuclear RNA-binding protein. Tenr is a 72-kDa protein that has one copy of a previously described RNA-binding domain. Northwestern blotting experiments showed that a maltose-binding protein-Tenr fusion binds to a variety of RNAs in vitro and that it does not bind to single-stranded or double-stranded DNA. The Tenr gene is transcribed exclusively in the testis, and its mRNA is restricted to cells from the pachytene spermatocyte stage through the round spermatid stage. Immunolocalization of the Tenr protein within the testis showed that it is first detected postmeiotically, demonstrating that the Tenr mRNA is under translational control. The Tenr protein is localized to round and early elongating spermatid cells, and confocal microscopy revealed a lattice-like nuclear distribution suggesting association with the nuclear scaffold. We suggest that the Tenr protein may be involved in testis-specific nuclear posttranscriptional processes such as heterogeneous nuclear RNA (hnRNA) packaging, alternative splicing, or nuclear/cytoplasmic transport of mRNAs.

Spnr, a murine RNA-binding protein that is localized to cytoplasmic microtubules.

Previous studies in transgenic mice have established the importance of the 3' untranslated region (UTR) of the spermatid-specific protamine-1 (Prm-1) mRNA in its translational control during male germ cell development. To clone genes that mediate the translational repression or activation of the Prm-1 mRNA, we screened cDNA expression libraries made with RNA from pachytene spermatocytes and round spermatids, with an RNA probe corresponding to the 3' UTR of Prm-1. We obtained six independent clones that encode Spnr, a spermatid perinuclear RNA-binding protein. Spnr is a 71-kD protein that contains two previously described RNA binding domains. The Spnr mRNA is expressed at high levels in the testis, ovary, and brain, and is present in multiple forms in those tissues. Immunolocalization of the Spnr protein within the testis shows that it is expressed exclusively in postmeiotic germ cells and that it is localized to the manchette, a spermatid-specific microtubular array. Although the Spnr protein is expressed too late to be directly involved in the translational repression of Prm-1 specifically, we suggest that the Spnr protein may be involved in other aspects of spermatid RNA metabolism, such as RNA transport or translational activation.

A primate model of Huntington's disease: functional neural transplantation and CT-guided stereotactic procedures.

In this article, we show that 1) computed tomographic (CT)-guided stereotactic infusion of an excitotoxin into the striatum of a nonhuman primate provides a useful neuropathologic and behavioral model for Huntington's disease. 2) High-resolution positron emission tomography (PET) can be used to image the decreased glucose utilization and the preservation of dopaminergic terminals in the lesioned striatum by using 2-fluoro-deoxy-D-glucose (2FDG) and N-(C-11)-methyl-2-beta-carbomethoxy-3-beta-phenyl tropane (CPT) as tracers. 3) Transplantation of cross-species striatal fetal tissue into the lesioned caudate-putamen reduces many of the abnormal motor movements and behavioral changes seen in the Huntington's disease primate model. 4) Graft rejection results in the return of the abnormal signs of the pregrafted state. These results indicate that treatment of the neuronal deficit in Huntington's disease can involve intervention at the local neuronal circuit level. CT-guided stereotactic implantation of cells that might protect or replace this defective circuitry may eventually provide an effective treatment for Huntington's disease.

Intracerebral implantation of nerve growth factor-producing fibroblasts protects striatum against neurotoxic levels of excitatory amino acids.

With the exception of L-DOPA pharmacological treatment in Parkinson's disease, the neurodegenerative diseases lack effective treatment. Previous studies of neurodegenerative diseases suggest that symptoms arise secondary to defects in local neuronal circuitry and cannot be treated effectively with systemic drug delivery. Therefore, a promising treatment is the application of fetal or genetically engineering cells which protect or replace neurons in deficient regions. Engineered cells can be derived from cell lines or grown from recipient host fibroblasts or other cells, then modified to produce and secrete substances at a specific area of the brain. A previous study using parallel intracerebral infusions of nerve growth factor and an excitotoxic amino acid into the rat striatum demonstrated a protective effect of nerve growth factor on neurons [Aloe L. (1987) Biotechnology 5, 1085-1086]. In order to further test this paradigm, we have utilized a biological delivery system of nerve growth factor by implanting fibroblasts into the rat striatum which secrete high levels of nerve growth factor, prior to infusing the neurotoxins quinolinate or quisqualate. Animals in this group had smaller lesions than did a group implanted with a similar non-nerve growth factor-producing graft. In addition, marked neuronal sparing was noted within areas of lesions in those animals containing a nerve growth factor-producing graft. These results indicate that implantation of genetically engineered nerve growth factor-secreting cells can be used to protect neurons at a specific target from excitotoxin-induced lesions.

Transmission and scanning electron microscopic study of chlordecone (Kepone) induced changes in the male mouse choroid plexus.

To investigate the effects of the estrogenic insecticide chlordecone on the morphology of mouse choroid plexus, 4 different doses (100.0, 250.0, 500.0 and 1000.0 micrograms) of the chemical were administered intraperitoneally to groups of adult males. Concurrent with the chlordecone treatments, another group of males received 10.0 micrograms estradiol-17 beta; the control group was treated with sesame oil vehicle. After 15 days of daily chemical injections, the mice were terminated and the choroid plexus from fourth ventricle examined morphologically. Scanning (SEM) and transmission (TEM) electron microscopic examination of control choroid plexus showed the cuboidal epithelium profusely covered with microvilli. SEM examinations of choroid plexus epithelium after chlordecone treatments revealed dose-dependent, cell alterations that effected the microvilli and cell surfaces. After the highest chlordecone dose, microvilli were no longer visible, the choroidal cell membrane appeared either smooth or pitted and there was evidence of increased luminal debris. TEM observations of the same choroid plexus cells revealed vacuolated cytoplasm, dilated endoplasmic reticulum, vacuolated mitochondria with disrupted cristae and cellular degeneration. SEM and TEM examination of choroid plexus after estradiol treatments revealed similar cellular alterations to those recorded after chlordecone treatments. The possible significance of these data are discussed.