Age-related decreases in GTP-cyclohydrolase-I immunoreactive neurons in the monkey and human substantia nigra.

Guanosine triphosphate cyclohydrolase I (GTPCHI) is a critical enzyme in catecholamine function and is rate limiting for the synthesis of the catecholamine co-factor tetrahydrobiopterin. The present study assessed the distribution of GTPCHI immunoreactivity (-ir) within the monkey and human ventral midbrain and determined whether its expression is altered as a function of age. Light and confocal microscopic analyses revealed that young monkeys and humans displayed GTPCHI-ir within melanin-containing and tyrosine-hydroxylase-ir neurons in primate substantia nigra. Stereological counts revealed that there was a 67.4% reduction in GTPCHI-ir neuronal number, a 63.5% reduction in GTPCHI-ir neuronal density, and a 37.6% reduction in neuronal volume in aged monkeys relative to young cohorts. Similar age-related changes were seen in humans, in whom there were significant reductions in the number of GTPCHI-ir nigral neurons in middle age (58.4%) and aged (81.5%) cases relative to young cohorts. The density of GTPCHI-ir neurons within the nigra was similarly reduced in middle-aged (63.0%) and aged (81.8%) cases. In contrast to monkeys, aged humans did not display shrinkage in the volume of GTPCHI-ir nigral neurons. The presence of numerous melanin-positive, but GTPCHI-ir immunonegative, neurons in the aged monkey and human nigra indicates that these decreases represent an age-related phenotypic downregulation of this enzyme and not a loss of neurons per se. These data indicate that there is a dramatic decrease in GTPCHI-ir in nonhuman primates and humans as a function of age and that loss of this enzyme may be partly responsible for the age-related decrease in dopaminergic tone within nigrostriatal systems.

Progress in direct striatal delivery of L-dopa via gene therapy for treatment of Parkinson's disease using recombinant adeno-associated viral vectors.

Viral vectors have recently been used successfully to transfer genes and express different proteins in the brain. This review discusses the requirements to consider human clinical trials in which recombinant adeno-associated virus vectors are used to transfer the genes necessary to produce l-dihydroxyphenylalanine (l-dopa) directly into the striatum of Parkinson's patients. Preclinical data that apply to the criteria defined as prerequisite for clinical trials are discussed. Thus, in animal models using recombinant adeno-associated virus vectors it has been demonstrated that l-dopa can be synthesized in the striatum after in vivo transduction. In addition, these l-dopa levels are sufficient to affect behavior in a dopamine-deficient animal model, the expression is extremely long-lasting, and the ability to transcriptionally regulate tyrosine hydroxylase has been demonstrated but not fully characterized. However, while immune responses to recombinant adeno-associated virus infection in the periphery have been studied, direct assessment of the potential immune response in the brain has not been sufficiently defined. Therefore, the rationale for delivering l-dopa directly to the striatum to treat Parkinson's disease is sound and the preclinical data are promising but all the issues surrounding this strategy are not resolved.

Long-term restoration of striatal L-aromatic amino acid decarboxylase activity using recombinant adeno-associated viral vector gene transfer in a rodent model of Parkinson's disease.

As a potential treatment for Parkinson's disease, viral vector-mediated over-expression of striatal L-aromatic amino acid decarboxylase was tested in an attempt to facilitate the production of therapeutic levels of dopamine after peripheral L-dihydroxyphenylalanine administration. The results of microdialysis and enzyme activity assays indicate that striatal decarboxylation of peripherally administered L-dihydroxyphenylalanine was enhanced by recombinant adeno-associated virus-mediated gene transfer of L-aromatic amino acid decarboxylase in unilateral 6-hydroxydopamine-lesioned rats. This gene transfer-induced increase in striatal decarboxylase activity was shown to remain undiminished over a six-month period and transgene expression was demonstrated to persist for at least one year. Unlike previous approaches involving delivery of either tyrosine hydroxylase, or tyrosine hydroxylase and L-aromatic amino acid decarboxylase transgenes together to accomplish unregulated dopamine delivery, the current study proposes a pro-drug strategy (peripheral L-dihydroxyphenylalanine administration after L-aromatic amino acid decarboxylase transduction). This strategy for dosage control could potentially allow lowered L-dihydroxyphenylalanine doses and potentially obviate complicated transcriptional regulation paradigms. These data suggest that the use of the non-pathogenic adeno-associated virus to transfer the L-aromatic amino acid decarboxylase gene into the striatum of Parkinson's disease patients may be an attractive gene therapy strategy.

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Nerve growth factor expressed in the medial septum following in vivo gene delivery using a recombinant adeno-associated viral vector protects cholinergic neurons from fimbria-fornix lesion-induced degeneration.

Nerve growth factor (NGF) has been shown to support the survival of axotomized medial septal cholinergic neurons after aspirative lesions of the fimbria-fornix (FF). This survival effect has been achieved utilizing intraventricular and intraparenchymal delivery of the NGF protein. While the use of NGF for the treatment of the cholinergic deficits present in Alzheimer's disease shows promise based on its efficacy in animal models, concerns about side-effects of intraventricular NGF delivery in humans have been raised. In the present study, NGF was delivered directly to the medial septum via a recombinant adeno-associated viral vector (rAAV) encoding the cDNA for human NGF prior to a FF lesion in rats. This rAAV-mediated NGF delivery was shown to significantly attenuate the medial septal cholinergic cell loss observed in animals receiving an equivalent injection of a control rAAV vector.

Recombinant adeno-associated viral vector-mediated glial cell line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after onset of progressive degeneration in a rat model of Parkinson's disease.

Previous work has demonstrated that viral vector mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF), when administered prior to a striatal injection of the specific neurotoxin, 6-hydroxydopamine (6-OHDA), can protect nigral dopamine (DA) neurons from cell death. When considering gene therapy for Parkinson's disease (PD), vector delivery prior to the onset of neuropathology is not possible and chronic delivery will likely be necessary in a GDNF-based PD therapy. The present study was undertaken to determine if GDNF delivered via a recombinant adeno-associated viral vector (rAAV) could affect nigral DA cell survival when initiated just after the administration of striatal 6-OHDA. The onset of rAAV-mediated GDNF transgene expression near the substantia nigra was determined to begin somewhere between 1 and 7 days after the 6-OHDA injection and subsequent vector administration. The cell survival data indicate that rAAV-GDNF delivery results in a highly significant sparing of nigral DA neurons. These data indicate that a single delivery of rAAV encoding GDNF is efficacious when delivered after the onset of progressive degeneration in a rat model of PD.

Regulation of gene expression in vivo following transduction by two separate rAAV vectors.

Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

In vivo L-DOPA production by genetically modified primary rat fibroblast or 9L gliosarcoma cell grafts via coexpression of GTPcyclohydrolase I with tyrosine hydroxylase.

To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinson's disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.

Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson's disease.

To achieve local, continuous L-DOPA delivery in the striatum by gene replacement as a model for a gene therapy for Parkinson's disease, the present studies used high titer purified recombinant adeno-associated virus (rAAV) containing cDNAs encoding human tyrosine hydroxylase (hTH) or human GTP-cyclohydrolase I [GTPCHI, the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis] or both to infect the 6-OHDA denervated rat striatum. Striatal TH and GTPCHI staining was observed 3 weeks after rAAV transduction, with little detectable perturbation of the tissue. Six months after intrastriatal rAAV transduction, TH staining was present but apparently reduced compared with the 3 week survival time. In a separate group of animals, striatal TH staining was demonstrated 1 year after rAAV transduction. Double staining studies using the neuronal marker NeuN indicated that >90% of rAAV-transduced cells expressing hTH were neurons. Microdialysis experiments indicated that only those lesioned animals that received the mixture of MD-TH and MD-GTPCHI vector displayed BH4 independent in vivo L-DOPA production (mean approximately 4-7 ng/ml). Rats that received the hTH rAAV vector alone produced measurable L-DOPA (mean approximately 1-4 ng/ml) only after receiving exogenous BH4. L-Aromatic amino acid decarboxylase blockade, but not 100 mM KCl-induced depolarization, enhanced L-DOPA overflow, and animals in the non-hTH groups (GTPCHI and alkaline phosphatase) yielded minimal L-DOPA. Although elevated L-DOPA was observed in animals that received mixed hTH and hGTPCHI rAAV vectors, there was no reduction of apomorphine-induced rotational behavior 3 weeks after intrastriatal vector injection. These data demonstrate that purified rAAV, a safe and nonpathogenic viral vector, mediates long-term striatal hTH transgene expression in neurons and can be used to successfully deliver L-DOPA to the striatum.

Practical aspects of the development of ex vivo and in vivo gene therapy for Parkinson's disease.

Current approaches to gene therapy of CNS disorders include grafting genetically modified autologous cells or introducing genetic material into cells in situ using a variety of viral or synthetic vectors to produce and deliver therapeutic substances to specific sites within the brain. Here we discuss issues related to the application of ex-vivo and in-vivo gene therapies as possible treatments for Parkinson's disease. Autologous monkey fibroblasts engineered ex-vivo to express tyrosine hydroxylase were grafted into MPTP-treated monkeys and found to express for up to 4 months. Adeno-associated (AAV) viral vectors expressing beta-galactosidase or tyrosine hydroxylase were introduced into monkey brains to determine the extent of infection and the types of cells infected by the vector at 21 days and 3 months. Gene expression was detected at both time points and was restricted to neurons in the striatum. These experiments demonstrate that two different approaches can be used to deliver proteins into the CNS. However, further technological advances are required to optimize gene delivery, regulation of gene expression, and testing in appropriate functional models before gene therapy can be considered for treating human disease.

Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats.

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson's disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-lacZ control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-lacZ-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the lacZ group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson's disease.

Maternal imprinting of human SNRPN, a gene deleted in Prader-Willi syndrome.

Prader-Willi syndrome (PWS), a human neuroendocrine disorder, is associated with deficiencies of paternal chromosome 15q12. Small nuclear ribonucleoprotein polypeptide N (SNRPN) is the first expressed gene identified in the PWS critically deleted region. Following our demonstration that the murine homologue of SNRPN is imprinted, we have characterized a sequence polymorphism within expressed portions of human SNRPN and show that human SNRPN is monoallelically expressed in fetal brain and heart and in adult brain. Analysis of maternal DNA and SNRPN cDNA confirmed that the maternal allele of SNRPN is not expressed in fetal brain and heart. Maternal imprinting of SNRPN supports the hypothesis that paternal absence of SNRPN is responsible for the PWS phenotype.

Regulation of tissue-specific splicing of the calcitonin/calcitonin gene-related peptide gene by RNA-binding proteins.

Transcripts of the rat calcitonin/calcitonin gene-related peptide (CGRP) gene are alternatively spliced in a tissue-specific manner resulting in the production of calcitonin mRNA and peptide in thyroid C cells and CGRP mRNA and peptide in neurons. Transfection studies using calcitonin and chimaeric human beta-globin/calcitonin exon minigene constructs showed that the splice acceptor and exon specific to calcitonin mRNA are spliced much less efficiently in CGRP-producing cells (F9 teratocarcinomas) than in cells that preferentially make calcitonin (HeLa cells). In vitro splicing of chimaeric human beta-globin/calcitonin transcripts in HeLa nuclear extracts were inhibited by the addition of nuclear extract from CGRP-favoring cells or tissues such as rat brain. This inhibition was specific as splicing of human beta-globin first intron transcripts was not affected by comparable amounts of rat brain extract. Fractionation of rat brain nuclear extracts allowed the partial purification of two brain-specific polypeptides of apparent molecular mass of 43 and 41 kDa which preferentially bind RNA containing the calcitonin-specific splice acceptor. Since these polypeptides cofractionate with the calcitonin mRNA-specific splicing inhibition activity, we suggest that they may mediate the inhibition of splicing observed in vitro and underlie, in part, the inefficient calcitonin mRNA production observed in CGRP-favoring cells in vivo.

Maternal imprinting of the mouse Snrpn gene and conserved linkage homology with the human Prader-Willi syndrome region.

Prader-Willi syndrome (PWS) is associated with paternal gene deficiencies in human chromosome 15q11-13, suggesting that PWS is caused by a deficiency in one or more maternally imprinted genes. We have now mapped a gene, Snrpn, encoding a brain-enriched small nuclear ribonucleoprotein (snRNP)-associated polypeptide SmN, to mouse chromosome 7 in a region of homology with human chromosome 15q11-13 and demonstrated that Snrpn is a maternally imprinted gene in mouse. These studies, in combination with the accompanying human mapping studies showing that SNRPN maps in the Prader-Willi critical region, identify SNRPN as a candidate gene involved in PWS and suggest that PWS may be caused, in part, by defects in mRNA processing.

A candidate mouse model for Prader-Willi syndrome which shows an absence of Snrpn expression.

The best examples of imprinting in humans are provided by the Angelman and Prader-Willi syndromes (AS and PWS) which are associated with maternal and paternal 15q11-13 deletions, respectively, and also with paternal and maternal disomy 15. The region of the deletions has homology with a central part of mouse chromosome 7, incompletely tested for imprinting effects. Here, we report that maternal duplication for this region causes a murine imprinting effect which may correspond to PWS. Paternal duplication was not associated with any detectable effect that might correspond with AS. Gene expression studies established that Snrpn is not expressed in mice with the maternal duplication and suggest that the closely-linked Gabrb-3 locus is not subject to imprinting. Finally, an additional new imprinting effect is described.

A tissue-specific enhancer in the rat-calcitonin/CGRP gene is active in both neural and endocrine cell types.

The calcitonin gene related peptide (CGRP) gene is a complex transcription unit that is expressed in a highly restricted pattern in both the nervous system, particularly in sensory ganglia and brainstem, and in the thyroid C cells of the endocrine system, with tissue-specific alternative RNA processing events generating transcripts encoding either the hormone, calcitonin, or the neuropeptide, CGRP. This pattern of expression in neural and endocrine tissues raises the question whether similar or distinct genomic elements are responsible for activation in both neural and endocrine cell types. We have identified a complex enhancer element, located more than 1 kilobase 5' of the transcription initiation site of the calcitonin/CGRP gene that functions in cells of neuronal or C cell origin, but not in any other cell type tested. At least two complementary regulatory sequences are required for the function of the cell-specific enhancer.

Induction of c-fos, calcitonin gene expression, and acidic fibroblast growth factor production in a multipeptide-secreting neuroendocrine cell line.

The multipeptide-secreting 44-2C cell line maintains differentiated function when grown in a serum-free, growth factor- and hormone-deprived milieu. The cells continue to synthesize and secrete calcitonin (CT), CT gene-related peptide, neurotensin, and somatostatin and respond to cellular secretagogues such as GRF and acidic and basic fibroblast growth factor. We designed experiments to ascertain the functional role(s) of cellular factors involved in the maintenance of the differentiated state in 44-2C cells. We report here the phenotypic transformation that occurs in these cells in the course of adjustment to the serum-free state. We also show the differential increase in CT-specific mRNA, the transient induction of c-fos, and the characterization of biologically active acidic fibroblast growth factor.

Calcium, dexamethasone, and the antiglucocorticoid RU-486 differentially regulate neuropeptide synthesis in a rat C cell line.

The differential regulation of neurotensin (NT), calcitonin (CT), and CT gene-related peptide (CGRP) production was studied in the clonal, rat C cell-derived, 44-2C cell line. Two experimental paradigms were used: cells were incubated with maximally effective concentrations of calcium (4.0 mM); alternatively, cells were treated with the synthetic glucocorticoid, dexamethasone (DEX). The specificity of the DEX-mediated response was assessed by using the synthetic antiglucocorticoid, RU-486. Calcium was not mitogenic in 44-2C cells and did not affect cell growth. Calcium increased the secretion and cellular accumulation of NT. In contrast, calcium treatment decreased CT content and release while it diminished the levels of CT- and CGRP-specific messenger RNA (mRNA) levels. DEX (10(-8) M) inhibited cell proliferation. NT content and secretion increased after DEX treatment, and this was potentiated by the addition of calcium. DEX-treated cells showed diminished CT content and secretion. The levels of CT- and CGRP-specific mRNA were significantly reduced in DEX-treated cultures. RU-486 antagonized the action of DEX and blocked DEX-inhibited cell proliferation. Inhibition of CT secretion by DEX was blocked by RU-486; CT- and CGRP-specific mRNA levels were increased in response to treatment with equimolar or 100-fold excess concentrations of RU-486. We conclude that NT secretion as well as CT/CGRP expression and release can be differentially regulated in the 44-2C cell line.

Splice commitment dictates neuron-specific alternative RNA processing in calcitonin/CGRP gene expression.

The calcitonin/CGRP gene is a complex transcription unit in which developmentally regulated cell-specific alternative RNA processing results in the production of CGRP mRNA in neurons, and of calcitonin mRNA in thyroid C cells. Alternative poly(A) site selection and exon splicing lead to the production of these alternative mature transcripts. The wild-type and a series of mutated calcitonin/CGRP genes were expressed in heterologous cell types, which produced predominantly calcitonin or CGRP mRNA. The results of these studies suggest that neurons express machinery or a factor that determines a specific alternative splicing pathway and consequently its associated alternative poly(A) site selection. It is hypothesized that a splice commitment regulatory factor might modulate pre-mRNA secondary structure, revealing a cryptic splice site required to generate CGRP mRNA in the brain.