Carl Rosenblad

Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat

Partial lesions of the nigrostriatal dopamine system have been investigated with respect to their ability to induce consistent long-lasting deficits in movement initiation and skilled forelimb use. In eight different lesion groups 6-hydroxydopamine (6-OHDA) was injected at one, two, three, or four sites into the lateral sector of the right striatum, in a total dose of 20-30 microgram. Impairments in movement initiation in a forelimb stepping test, and in skilled paw use in a paw-reaching test, was seen only in animals where the severity of the lesion exceeded a critical threshold, which was different for the different tests used: single (1 x 20 microgram) or two-site (2 x 10 microgram) injections into the striatum had only small affects on forelimb stepping, no effect on skilled paw use. More pronounced deficits were obtained in animals where the same total dose of 6-OHDA was distributed over three or four sites along the rostro-caudal extent of the lateral striatum or where the injections were made close to the junction of the globus pallidus. The results show that a 60-70% reduction in tyrosine hydroxylase (TH)-positive fiber density in the lateral striatum, accompanied by a 50-60% reduction in TH-positive cells in substantia nigra (SN), is sufficient for the induction of significant impairment in initiation of stepping. Impaired skilled paw-use, on the other hand, was obtained only with a four-site (4 x 7 microgram) lesion, which induced 80-95% reduction in TH fiber density throughout the rostrocaudal extent of the lateral striatum and a 75% loss of TH-positive neurons in SN. Drug-induced rotation, by contrast, was observed also in animals with more restricted presymptomatic lesions. The results indicate that the four-site intrastriatal 6-OHDA lesion may be a relevant model of the neuropathology seen in parkinsonian patients in a manifest symptomatic stage of the disease and may be particularly useful experimentally since it leaves a significant portion of the nigrostriatal projection intact which can serve as a substrate for regeneration and functional recovery in response to growth promoting and neuroprotective agents.

Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system.

Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human α-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including α-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinsons disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30–80% of the nigral dopamine neurons, a 40–50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50–60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that α-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant α-synuclein, pointing to a key role for α-synuclein in the pathogenesis of Parkinsons disease. Targeted overexpression of α-synuclein in the nigrostriatal system may provide a new animal model of Parkinsons disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.

Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model

During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinsons disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3-6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinsons disease.

Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson’s disease after administration into the striatum or the lateral ventricle

Both glial cell line‐derived neurotrophic factor (GDNF) and its recently discovered congener, neurturin (NTN), have been shown to excert neuroprotective effects on lesioned nigral dopamine (DA) neurons when administered at the level of the substantia nigra. In the present study, we have explored the relative in vivo potency of these two neurotrophic factors using two alternative routes of administration, into the striatum or the lateral ventricle, which may be more relevant in a clinical setting. In rats subjected to an intrastriatal (IS) 6‐hydroxydopamine (6‐OHDA) lesion, GDNF and NTN were injected every third day for 3 weeks starting on the day after the 6‐OHDA injection. GDNF provided almost complete (90–92%) protection of the lesioned nigral DA neurons after both IS and intracerebroventricular (ICV) administration. NTN, by contrast, was only partially effective after IS injection (72% sparing) and totally ineffective after ICV injection. Although the trophic factor injections protected the nigral neurons from lesion‐induced cell death, the level of expression of the phenotypic marker, tyrosine hydroxylase (TH), was markedly reduced in the rescued cell bodies. The extent of 6‐OHDA‐induced DA denervation in the striatum was unaffected by both types of treatment; consistent with this observation, the high rate of amphetamine‐induced turning seen in the lesioned control animals was unaltered by either GDNF or NTN treatment. In the GDNF‐treated animals, and to a lesser extent also after IS NTN treatment, prominent axonal sprouting was observed within the globus pallidus, at the level where the lesioned nigrostriatal axons are known to end at the time of onset of the neurotrophic factor treatment. The results show that GDNF is highly effective as a neuroprotective and axon growth‐stimulating agent in the IS 6‐OHDA lesion model after both IS and ICV administration. The lower efficacy of NTN after IS, and particularly ICV, administration may be explained by the poor solubility and diffusion properties at neutral pH.

Studies on neuroprotective and regenerative effects of GDNF in a partial lesion model of Parkinson's disease

Intrastriatal 6-hydroxydopamine injections in rats induce partial lesions of the nigrostriatal dopamine (DA) system which are accompanied by a delayed and protracted degeneration of DA neurons within the substantia nigra. By careful selection of the dose and placement of the toxin it is possible to obtain reproducible and regionally defined partial lesions which are well correlated with stable functional deficits, not only in drug-induced behaviors but also in spontaneous motoric and sensorimotoric function, which are analogous to the symptoms seen in patients during early stages of Parkinsons disease. The intrastriatal partial lesion model has proved to be particularly useful for studies on the mechanisms of action of neurotrophic factors since it offers opportunities to investigate both protection of degenerating DA neurons during the acute phases after the lesion and stimulation of regeneration and functional recovery during the chronic phase of the postlesion period when a subset of the spared nigral DA neurons persist in an atrophic and dysfunctional state. In the in vivo experiments performed in this model glial cell line-derived neurotrophic factor (GDNF) has been shown to exert neurotrophic effects both at the level of the cell bodies in the substantia nigra and at the level of the axon terminals in the striatum. Intrastriatal administration of GDNF appears to be a particularly effective site for induction of axonal sprouting and regeneration accompanied by recovery of spontaneous sensorimotor behaviors in the chronically lesioned nigrostriatal dopamine system.

Spatial learning impairments in rats with selective immunolesion of the forebrain cholinergic system

A monoclonal antibody to the low-affinity NGF receptor, 192 IgG, coupled to a cytotoxin, saporin, was recently introduced as an efficient selective neurotoxin for the NGFr-bearing cholinergic neurones in the rat basal forebrain. In the present study we report that an intracerebroventricular injection of this 192 IgG-saporin conjugate induces a severe, long-lasting spatial learning impairment, as assessed in the Morris water-maze task. This behavioural impairment was associated with 65-90% depletion of choline acetyltransferase activity (ChAT) in the hippocampus and cortex. ChAT activity associated with other cholinergic neurone systems in the brain (striatum, mesencephalon, spinal cord), was left virtually unaffected. This new immunotoxin holds great promise as a tool for selective and efficient lesions of the forebrain cholinergic system in functional and behavioural studies.

Preservation of a functional nigrostriatal dopamine pathway by GDNF in the intrastriatal 6-OHDA lesion model depends on the site of administration of the trophic factor

Here we studied whether glial cell line‐derived neurotrophic factor (GDNF), given as a single bolus injection before an intrastriatal 6‐hydroxydopamine (6‐OHDA) lesion, can protect the nigrostriatal dopamine neurons against the toxin‐induced damage and preserve normal motor functions in the lesioned animals. GDNF or vehicle was injected in the striatum (25 μg), substantia nigra (25 μg) or lateral ventricle (50 μg) 6 h before the 6‐OHDA lesion (20 μg/3 μL). Motor function was evaluated by the stepping and drug‐induced motor asymmetry tests. Lesioned animals given vehicle alone showed a clear ipsilateral‐side bias in response to amphetamine (13 turns/min), a moderate contralateral‐side bias to apomorphine (4.5 turns/min) and a moderate to severe stepping deficit on the contralateral forepaw (three to four steps, as compared with 11–13 steps on the unimpaired side). Injection of GDNF into the striatum had a significant protective effect both on nigrostriatal function (1–2 turns/min in the rotation tests and seven to eight steps in the stepping test), and the integrity of the nigrostriatal pathway, seen as a protection of both the cell bodies in the substantia nigra and the dopamine innervation in the striatum. Injection of GDNF in the nigra had a protective effect on the nigral cell bodies, but not the striatal innervation, and failed to provide any functional benefit. In contrast, intranigral GDNF had deleterious effects on both the striatal TH‐positive fibre density and on drug‐induced rotation tests. Intraventricular injection had no effect. We conclude that preservation of normal motor functions in the intrastriatal 6‐OHDA lesion model requires protection of striatal terminal innervation, and that this can be achieved by intrastriatal, but not nigral or intraventricular, administration of GDNF.

In vivo protection of nigral dopamine neurons by lentiviral gene transfer of the novel GDNF-family member neublastin/artemin.

The glial cell line-derived neurotrophic factor (GDNF)-family of neurotrophic factors consisted until recently of three members, GDNF, neurturin, and persephin. We describe here the cloning of a new GDNF-family member, neublastin (NBN), identical to artemin (ART), recently published (Baloh et al., 1998). Addition of NBN/ART to cultures of fetal mesencephalic dopamine (DA) neurons increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons by approximately 70%, similar to the maximal effect obtained with GDNF. To investigate the neuroprotective effects in vivo, lentiviral vectors carrying the cDNA for NBN/ART or GDNF were injected into the striatum and ventral midbrain. Three weeks after an intrastriatal 6-hydroxydopamine lesion only about 20% of the nigral DA neurons were left in the control group, while 80-90% of the DA neurons remained in the NBN/ART and GDNF treatment groups, and the striatal TH-immunoreactive innervation was partly spared. We conclude that NBN/ART, similarly to GDNF, is a potent neuroprotective factor for the nigrostriatal DA neurons in vivo.

Sequential administration of GDNF into the substantia nigra and striatum promotes dopamine neuron survival and axonal sprouting but not striatal reinnervation or functional recovery in the partial 6-OHDA lesion model

Glial cell line-derived neurotrophic factor (GDNF) has prominent survival-promoting effects on lesioned nigrostriatal dopamine neurons, but understanding of the conditions under which functional recovery can be obtained remains to be acquired. We report here the time course of nigrostriatal axon degeneration in the partial lesion model of Parkinsons disease and the morphological and functional effects of sequential administration of GDNF in the substantia nigra (SN) and striatum during the first 5 weeks postlesion. By 1 day postlesion, the nigrostriatal axons had retracted back to the level of the caudal globus pallidus. Over the next 6 days axonal retraction progressed down to the SN, and during the following 7 weeks 74% of tyrosine hydroxylase-positive (TH(+)) and 84% of retrogradely labeled nigral neurons were lost, with a more pronounced loss in the rostral part of the SN. GDNF administration protected 70 and 72% of the nigral TH(+) and retrogradely labeled cell bodies, respectively, but did not prevent the die-back of the lesioned nigrostriatal axons. Although clear signs of sprouting were observed close to the injection site in the striatum as well as in the globus pallidus, the overall DA innervation of the striatum [as measured by [(3)H]-N-[1-(2-benzo(b)thiopenyl)cyclohexyl]piperidine-binding autoradiography] was not improved by the GDNF treatment. Moreover, the lesion-induced deficits in forelimb akinesia and drug-induced rotation were not attenuated. We conclude that functional recovery in the partial lesion model depends not only on preservation of the nigral cell bodies, but more critically on the ability of GDNF to promote significant reinnervation of the denervated striatum.

Delayed infusion of GDNF promotes recovery of motor function in the partial lesion model of Parkinson's disease

Here we studied the effects of glial cell line‐derived neurotrophic factor (GDNF) in a rat model that represents the symptomatic stages of Parkinsons disease. GDNF was infused starting 2 weeks after an intrastriatal 6‐hydroxydopamine (6‐OHDA) lesion in order to halt the ongoing degeneration of the nigrostriatal dopaminergic neurons. GDNF or vehicle was infused in the striatum or the lateral ventricle via an osmotic minipump over a total 4‐week period (2–6 weeks postlesion). Motor function was evaluated by the stepping, paw reaching and drug‐induced motor asymmetry tests before the pump infusion was initiated, and was repeated once during (5 weeks postlesion) and twice after the withdrawal of the minipumps (7 and 11 weeks postlesion). We found that within two weeks following the lesion ≈ 40% of the nigral TH‐positive neurons were lost. In the vehicle infusion groups there was an additional 20% cell loss between 2 and 12 weeks after the lesion. This latter cell loss occurred mainly in the caudal part of the SN whereas the cell loss in the rostral SN was almost complete within the first two weeks. Ventricular GDNF infusion completely blocked the late degenerating neurons in the caudal SN and had long lasting behavioural effects on the stepping test and amphetamine rotation, extending to 6 weeks after withdrawal of the factor. Striatal infusion affected the motor behaviour transiently during the infusion period but the motor performance of these animals returned to baseline upon cessation of the GDNF delivery, and the delayed nigral cell loss was marginally affected. We conclude that intraventricular GDNF can successfully block the already initiated degenerative process in the substantia nigra, and that the effects achieved via the striatal route, when GDNF is given acutely after the lesion, diminish as the fibre terminal degeneration proceeds.