Anne D. Zurn

Isolation of Multipotent Neural Precursors Residing in the Cortex of the Adult Human Brain

Multipotent precursors able to generate neurons, astrocytes, and oligodendrocytes have previously been isolated from human brain embryos and recently from neurogenic regions of the adult human brains. The isolation of multipotent neural precursors from adult human should open new perspectives to study adult neurogenesis and for brain repair. The present study describes the in vitro isolation from adult human brains of a progenitor responsive to both epidermal and basic fibroblast growth factors that forms spheres as it proliferates. Single spheres derived from various regions of the brain generate in vitro neurons, astrocytes, and oligodendrocytes. The clonal origin of the spheres was revealed by genomic viral insertion using lentiviral vector. Interestingly, this vector appears to be a potent tool for gene transfer into human neural progeny. Ninety-six percent of the spheres investigated were multipotent. Multipotent precursors were isolated from all brain regions studied, including the temporal and the frontal cortex, the amygdala, the hippocampus, and the ventricular zone. This study is the first evidence that primitive precursors such as multipotent precursors exist in the adult human cortex and can reside far from the ventricles. Neurogenesis derived from adult human progenitors differ to murine neurogenesis by the requirement of laminin for oligodendrocyte generation and by the action of basic-fibroblast growth factor and platelet derived growth factor that prevented the formation of oligodendrocytes and neurons. Moreover, the differentiation of human adult precursors seems to differ from fetal ones: adult precursors do not necessitate the removal of mitogen for differentiation. These results indicate that the study of adult multipotent precursors is a new platform to study adult human neurogenesis, potentially generate neural cells for transplantation, and design protocols for in vivo stimulation.

Self-Inactivating Lentiviral Vectors with Enhanced Transgene Expression as Potential Gene Transfer System in Parkinson's Disease

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinsons disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

Lentiviral vectors as a gene delivery system in the mouse midbrain : Cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF

Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinsons disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinsons disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinsons disease.

The Copper Chelator d-Penicillamine Delays Onset of Disease and Extends Survival in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

A subpopulation of familial cases of amyotrophic lateral sclerosis has been linked to mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). There is in vitro evidence that certain SOD1 mutants, in addition to their normal dismutation function, show increased ability of the enzyme to act as a peroxidase. This reaction is sensitive to inhibition by copper chelators. To test this hypothesis in vivo, we administered the copper chelator d‐penicillamine to a transgenic mouse model of familial amyotrophic lateral sclerosis overexpressing a mutated form of human SOD1. We demonstrate that oral administration of d‐penicillamine is able to delay the onset of the disease and extend the survival of these mice. Histological studies also showed a decreased loss of facial motor neurons in d‐penicillamine‐treated transgenic mice, corroborating the slower evolution of the disease in these animals. These results suggest that copper chelators may benefit patients with familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene.

GDNF Reduces Drug-Induced Rotational Behavior after Medial Forebrain Bundle Transection by a Mechanism Not Involving Striatal Dopamine

Parkinson’s disease (PD) is characterized by the progressive loss of the substantia nigra (SN) dopaminergic neurons projecting to the striatum. Neurotrophic factors may have the potential to prevent or slow down the degenerative process occurring in PD. To that end, we examined whether low amounts of glial cell line-derived neurotrophic factor (GDNF) continuously released from polymer-encapsulated genetically engineered cells are able to prevent the loss of tyrosine hydroxylase immunoreactivity (TH-IR) in SN neurons and ameliorate the amphetamine-induced rotational asymmetry in rats that have been subjected to a unilateral medial forebrain bundle (MFB) axotomy. Baby hamster kidney (BHK) cells transfected with the cDNA for GDNF were encapsulated in a polymer fiber and implanted unilaterally at a location lateral to the MFB and rostral to the SN. ELISA assays before implantation show that the capsules release ∼5 ng of GDNF/capsule per day. One week later, the MFB was axotomized unilaterally ipsilateral to the capsule placement. Seven days later, the animals were tested for amphetamine-induced rotational asymmetry and killed. The striatum was excised and analyzed either for catecholamine content or TH-IR, while the SN was immunostained for the presence of TH-IR. GDNF did not prevent the loss of dopamine in the striatum. However, GDNF significantly rescued TH-IR neurons in the SN pars compacta. Furthermore, GDNF also significantly reduced the number of turns per minute ipsilateral to the lesion under the influence of amphetamine. Improvement of rotational behavior in the absence of dopaminergic striatal reinnervation may reflect neuronal plasticity in the SN, as suggested by the dendritic sprouting observed in animals receiving GDNF. These results illustrate that the continuous release of low levels of GDNF close to the SN is capable of protecting the nigral dopaminergic neurons from an axotomy-induced lesion and significantly improving pharmacological rotational behavior by a mechanism other than dopaminergic striatal reinnervation.

Glial cell line-derived neurotrophic factor (GDNF), a new neurotrophic factor for motoneurones

GLIAL cell line-derived neurotrophic factor (GDNF) has been postulated to be a specific dopaminergic neurotrophic factor since it selectively enhances the survival of dopaminergic neurones in vitro. We report here that GDNF can also act as a neurotrophic factor for motoneurones. GDNF released by GDNF-transfected BHK cells increases the activity of choline acetyltransferase (ChAT) in cultures from embryonic rat ventral mesencephalon containing cholinergic neurones from cranial motor nuclei, and in cultured spinal motoneurones. Furthermore, local application of polymer-encapsulated BHK cells releasing GDNF to transected facial nerve in newborn rats diminishes the death of motoneurones normally occurring after axotomy in the neonatal period. The present results indicate that GDNF may have a therapeutic potential in human motoneurone diseases such as amyotrophic lateral sclerosis.

Neuronal GDNF Expression in the Adult Rat Nervous System Identified By In Situ Hybridization

Glial cell line‐derived neurotrophic factor (GDNF) is a potent neurotrophic factor which has been purified on the basis of its ability to promote the survival of dopaminergic neurons in vitro. GDNF has subsequently been cloned and its sequence shown to be distantly related to transforming growth factor‐β (TGF‐β). To identify GDNF expressing cells in the adult rat brain, in situ hybridization using a digoxygenin (DIG)‐labelled riboprobe has been performed. Our results show that GDNF mRNA is mainly expressed in neurons and that its synthesis is not restricted to dopaminergic areas. It is widely expressed in the cortex, the hippocampus, the striatum, the substantia nigra, the thalamus, the cerebellum and the spinal cord. Neuronal GDNF expression varies among brain regions as determined by the intensity of the in situ signal. Double labelling of the substantia nigra using tyrosine hydroxylase immunohistochemistry, associated with GDNF in situ hybridization, show that the majority of dopaminergic neurons express GDNF. The widespread expression of GDNF throughout the adult brain suggests that its administration in Parkinsons disease should be restricted to the altered structures, in order to avoid possible deleterious side effects.

Combined effects of GDNF, BDNF, and CNTF on motoneuron differentiation in vitro

We have previously shown that glial cell line‐derived neurotrophic factor (GDNF), in addition to promoting the survival of dopaminergic neurons in cultures from embryonic rat ventral mesencephalon, also increases the activity of choline acetyltransferase (ChAT) in the cranial motoneurons present in these cultures (Zurn et al.: Neuroreport 6:113–118, 1994). By using the intermediate filament protein peripherin as a motoneuron marker, we report here that GDNF increases the number of motoneurons as well as the length of their neurites. Brain‐derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) also promote ChAT activity, motoneuron survival, and neurite outgrowth in these cultures, but to varying degrees. Although these three molecules have similar effects on cultured motoneurons, we provide evidence for a distinct mode of action of GDNF, BDNF, and CNTF, since combinations of GDNF and BDNF, GDNF and CNTF, and BDNF and CNTF have either additive or synergistic effects on ChAT activity and motoneuron number. In addition to the previously described motoneuron‐specific neurotrophic factors BDNF and CNTF, GDNF combined with the latter two factors may provide an important tool for the treatment of human motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy, both by increasing efficiency of treatment, and by decreasing the likelihood of deleterious side‐effects.

Glial cell line-derived neurotrophic factor released by synthetic guidance channels promotes facial nerve regeneration in the rat

Regeneration of the human facial nerve after lesion is often limited, leading to severe functional impairments, in particular when repair is delayed for several months, when cross‐facial nerve grafts have to be performed, or in elderly patients. To improve the outcome, the potential accelerating and maturating effects of the neurotrophic factors glial cell line‐derived neurotrophic factor (GDNF) and neurotrophin‐3 (NT‐3) on nerve regeneration were assessed using an axotomy model of the rat facial nerve. One‐centimeter‐long synthetic guidance channels releasing the neurotrophic factors over several weeks were used to bridge an 8 mm nerve gap, a distance that does not allow regeneration in the absence of growth factors. Nerve cables regenerated in the presence of GDNF showed a large number of myelinated axons 6 weeks after grafting (871 ± 373, n = 5), whereas only 106 ± 86 (n = 5) myelinated axons were counted in the presence of NT‐3. Retrograde labeling with fluorogold revealed 981 ± 450 (n = 5) and 53 ± 38 (n = 5) retrogradely labeled motoneurons in the facial nucleus in the presence of GDNF and NT‐3, respectively. No regenerated axons or retrogradely labeled cells were observed in the absence of growth factors (n = 6). These results demonstrate that GDNF, as previously described for the sciatic nerve, a mixed sensory and motor nerve, is also very efficient in promoting regeneration of the facial nerve, an essentially pure motor nerve. GDNF may therefore be useful in improving facial nerve regeneration in the clinic.

Nerve growth factor- and neurotrophin-3-releasing guidance channels promote regeneration of the transected rat dorsal root.

Dorsal roots have a limited regeneration capacity after transection. To improve nerve regeneration, the growth-promoting effects of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) were evaluated. The proteins were continuously released by synthetic nerve guidance channels bridging a 4-mm gap in the transected dorsal root. Four weeks after lesion, the regenerated nerve cables were analyzed for the presence of myelinated and unmyelinated axons. While BDNF showed a limited effect on axonal regeneration (863 +/- 39 axons/regenerated nerve, n = 6), NGF (1843 +/- 482) and NT-3 (1495 +/- 449) powerfully promoted regeneration of myelinated axons compared to channels releasing the control protein bovine serum albumin (293 +/- 39). In addition, NGF, but not BDNF nor NT-3, had a potent effect on the regeneration of unmyelinated axons (NGF, 55 +/- 1.4; BDNF, 4 +/- 0.3; NT-3, 4.7 +/- 0.3 axons/100 microm(2); n = 6). The present study suggests that synthetic nerve guidance channels slowly and continuously releasing the neurotrophins NGF and NT-3 can overcome the limited regeneration of transected dorsal root.