3.1. Effects of GDNF in vivo
In normal adult rats, a single injection of GDNF into either the substantia nigra or striatum significantly increased the levels of dopamine and its metabolites in the striatum and nigra (Martin et al., 1996). Several studies have reported neuroprotective and functional (improvement of motor symptoms) effects of GDNF in adult animal models of PD (for reviews, see Balemans & Van Hul, 2002; Bjorklund et al., 2009; Bjorklund & Lindvall, 2000; Bjorklund et al., 1997; Deierborg et al., 2008; Gash et al., 1996; Ramaswamy et al., 2009). In one early study, repeated injections of recombinant rat GDNF protected against dopaminergic cell loss induced by transection of the adult rat medial forebrain bundle (MFB), the fibre bundle containing the dopaminergic projections from the substantia nigra to the striatum (Beck et al., 1995). Another study found that delivery of GDNF using polymer-encapsulated baby hamster kidney (BHK) cells at one week before axotomy of the adult rat MFB induced motor improvements and rescued nigral dopaminergic neurones, without significant protection of striatal dopamine levels (Tseng et al., 1997).
The most widely-used laboratory model of PD involves unilateral injection of the selective dopaminergic toxin, 6-OHDA, into either the MFB, the substantia nigra or the striatum of the adult rat. This results in the degeneration of nigrostriatal dopaminergic neurones and consequent depletion of striatal dopamine transmission on one side of the brain. Stereotaxic injection of 6-OHDA into the MFB or substantia nigra induces a substantial lesion of the nigrostriatal pathway (Ungerstedt & Arbuthnott, 1970), while intrastriatal injection of this toxin induces a more protracted neurodegeneration, which begins after a delay of about a week and continues for several weeks (Sauer & Oertel, 1994). Several groups have examined the effects of intracerebral injection of recombinant GDNF in rats with 6-OHDA-induced lesions of the MFB. A single dose of GDNF to the substantia nigra, given four weeks after a 6-OHDA lesion of MFB, resulted in improvements in motor deficits (drug-induced rotational asymmetry, a commonly-used motor test in rat PD models) and preservation of nigral dopaminergic neurones (Bowenkamp et al., 1995; Hoffer et al., 1994). GDNF injection just above the substantia nigra and into the lateral ventricle immediately before 6-OHDA injection into the MFB conferred significant protective effects on nigral dopaminergic neurones, on striatal dopamine release and uptake, and on motor function (Opacka-Juffry et al., 1995). In adult rats with bilateral 6-OHDA lesions of the MFB, a model of severe PD, injection of high doses of GDNF into the lateral ventricles resulted in improved motor function and sparing of nigral dopaminergic neurones (Bowenkamp et al., 1997). Although GDNF has been reported to have protective actions in 6-OHDA-lesioned rats of all ages, its effects appear to be dependent on the age of the host, as one study found that young rats displayed significantly higher levels of neuroprotection than aged rats (Fox et al., 2001). This may be relevant to clinical trials, where the age of the patient may determine the extent of neuroprotection that is achievable with GDNF treatment.
The intrastriatal lesion model has been used extensively in studies on neurotrophic factors, since in this model it is possible to administer the factors while the progressive neurodegeneration is still taking place. Supranigral administration of recombinant human GDNF for four weeks, starting at the day of a 6-OHDA-induced lesion of the rat striatum, completely prevented nigral cell death (Sauer et al., 1995). When administered one day before a 6-OHDA lesion of either the striatum or substantia nigra, intranigral injection of GDNF had protective effects on rat nigral dopaminergic cell bodies (Kearns & Gash, 1995). A series of four intrastriatal injections of GDNF was found to decrease drug-induced rotations and preserve nigrostriatal dopaminergic neurones in adult rats with 6-OHDA-induced striatal lesions (Shults et al., 1996). Long-term rescue of nigrostriatal dopaminergic neurones from intrastriatal 6-OHDA lesions was reported after short-term GDNF treatment, beginning five days after the lesion and being administered every fourth day for one month (Winkler et al., 1996). Long-term protection against rotational asymmetry, reductions in striatal dopamine levels and uptake, and death of nigral dopaminergic cell bodies induced by 6-OHDA lesions of the MFB was conferred by a single dose of GDNF, divided between the lateral ventricle and substantia nigra (Sullivan et al., 1998). GDNF injections into the striatum at one week after an intrastriatal 6-OHDA lesion resulted in reinnervation of the striatum as well as recovery of motor function (Rosenblad et al., 1998), indicating that the ability of intrastriatal GDNF injection to confer behavioural improvements may be due to its effects on the remaining striatal afferents in the partially-denervated striatum.
For application to clinical studies, the optimal injection site for production of safe and effective results is obviously an important consideration. Some studies have directly compared the sites of administration of GDNF in 6-OHDA-lesioned rats. In a study designed to compare the various sites of intracerebral injection, intrastriatal GDNF had significant protective effects on motor function and on the integrity of the nigrostriatal pathway, intranigral GDNF protected nigral cell bodies but not striatal innervation or motor function, while intraventricular GDNF had no significant effects (Kirik et al., 2000). Another study by the same group found that delayed intraventricular infusion of GDNF starting two weeks after an intrastriatal 6-OHDA lesion had profound protective effects on the integrity and function of the nigrostriatal pathway, which lasted for six weeks after cessation of GDNF infusion, whereas the effects of intrastriatal infusion stopped upon withdrawal of GDNF (Kirik et al., 2001). Another group found that delayed intrastriatal infusion of a high dose of GDNF at four weeks after an intrastriatal 6-OHDA lesion induced restorative effects on the nigrostriatal dopaminergic pathway, in terms of both motor behaviour and the integrity of dopaminergic neurones and their terminals (Aoi et al., 2000). Thus, the intrastriatal route of administration appears to be the most efficacious in this rodent model of PD. In another study, sequential application of GDNF over the nigra for two weeks, followed by injections of GDNF into the striatum for three weeks, in rats with intrastriatal 6-OHDA lesions, protected nigral dopaminergic cell bodies but did not prevent striatal denervation or improve motor function (Rosenblad et al., 2000). This suggests that the motor improvements observed in the other studies were dependent on an ability of GDNF to induce reinnervation of the lesioned striatum, perhaps by stimulating axonal sprouting from the remaining dopaminergic neurones. Thus, once the axonal retraction to the level of the nigra has occurred, application of GDNF to the striatum appears to be ineffective. This is an important consideration for clinical studies, as it suggests that there is a window of opportunity in which GDNF application may be therapeutically effective, but that this factor may not be useful at advanced stages of the disease.
In another commonly-used animal model of PD, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated adult mice, GDNF administration induces potent effects on nigrostriatal dopaminergic neurones (Tomac et al., 1995). This study found that GDNF could be injected either before or one week after MPTP treatment, to confer significant preventative or restorative effects, respectively. Another study reported that GDNF injection into the striatum at one week after MPTP treatment of young or aged mice induced recovery of motor function (Date et al., 1998).
Another well-established animal model of PD is the MPTP-treated non-human primate brain, which has the advantage of being closer than the rodent brain to the human brain in terms of organisation and size. Gash and colleagues showed that administration of recombinant GDNF every four weeks in MPTP-treated rhesus monkeys induced motor recovery as well as a significant increase in the size of cell nigral dopaminergic neurones and in the density of their striatal projections (Gash et al., 1996). Combined application of oral levodopa and intracerebroventricular GDNF resulted in significant behavioural improvements with reduced levodopa-induced side-effects, in MPTP-treated rhesus monkeys (Miyoshi et al., 1997). Another study found that intraventricular GDNF improved motor function and reduced levodopa-induced dyskinesias in MPTP-treated monkeys (Iravani et al., 2001). Significant recovery of motor function for at least four months was reported in MPTP-treated monkeys that had received GDNF into the lateral ventricles (Zhang et al., 1997). Intraventricular injections of GDNF were found to induce increases in dopamine levels in the substantia nigra, but not the striatum, of MPTP-treated rhesus monkeys (Gerhardt et al., 1999). Infusion of GDNF into the putamen of MPTP-treated rhesus monkeys induced a gradual and significant reduction in parkinsonian symptoms (Grondin et al., 2002). This appeared to be a regenerative action, since GDNF was injected at three months after the MPTP lesion, when the nigrostriatal pathway had presumably undergone significant degeneration. Chronic intraputaminal administration of GDNF in aged rhesus monkeys had a long-lasting protective action on nigrostriatal dopaminergic neurones and on motor function, without any adverse side-effects (Maswood et al., 2002). In addition to effects on PD models, a single intranigral injection of GDNF can induce increases in nigrostriatal dopamine neurotransmission in normal rhesus monkeys, lasting for at least three weeks (Gash et al., 1995).
Like all neurotrophic proteins, GDNF is metabolised rapidly in the brain and thus single injections of this factor cannot confer permanent effects. Gene therapy approaches have been applied in attempts to achieve long-term and targeted delivery of GDNF to the injured nigrostriatal pathway. An adenoviral vector was used to deliver GDNF into or close to the substantia nigra (Choi-Lundberg et al., 1997; Mandel et al., 1997) or into the striatum (Balemans & Van Hul, 2002; Bilang-Bleuel et al., 1997; Choi-Lundberg et al., 1998) of rats with intrastriatal 6-OHDA lesions. Each of these studies reported significant motor improvements, as well as direct effects on nigral dopaminergic neurones and their terminals. When administered into the substantia nigra at ten weeks after a 6-OHDA lesion of the adult rat MFB, adenoviral vector-delivered GDNF induced significant motor improvements and recovery of nigrostriatal dopamine neuronal function (Lapchak et al., 1997). Adenoviral-delivered GDNF induced behavioural and neuroprotective effects when injected into the substantia nigra, but not when injected intrastriatally, in rats that had intrastriatal 6-OHDA lesions (Kozlowski et al., 2000). In MPTP-treated mice, adenoviral vector-mediated GDNF delivery to the striatum prevented depletion of striatal dopamine levels (Kojima et al., 1997). A study which compared the effects of intrastriatal and perinigral injection of an adenoviral vector encoding GDNF found that, while both injection routes conferred protective effects on dopaminergic cell bodies in the nigra, only the intrastriatal route reduced motor deficits in rats with intrastriatal 6-OHDA lesions (Connor et al., 1999).
Another vector system, based on adeno-associated virus (AAV) 2, has also shown efficacy in animal models of PD. Mandel and co-workers reported significant protective effects on the nigrostriatal pathway and its functioning, in adult rats with intrastriatal 6-OHDA lesions following intranigral injection of AAV2-GDNF either three weeks before (Mandel et al., 1997) or just after the lesion (Mandel et al., 1999). AAV2-mediated delivery of GDNF to the striatum, but not to the substantia nigra, induced gradual behavioural recovery and regeneration of the 6-OHDA-lesioned nigrostriatal system in adult rats (Kirik et al., 2000). AAV vectors have advantages over adenoviral vectors in that they can integrate and stably express their transgene product in non-dividing cells such as neurons. Also, they are relatively safe as there is little or no host immune response, due to the absence of viral genes in these vectors. Their disadvantage is that they can only deliver gene constructs of relatively small size compared to those that adenoviral vectors can accommodate. Furthermore, there is a delay before the transgene is expressed following intracerebral injection of an AAV vector.
A third type of vector system, based on lentiviruses, has also been used to deliver GDNF in PD animal models, with promising results. Lentiviral vectors have the capacity to deliver large transgenes and they can integrate efficiently into non-dividing cells. Delivery of the human GDNF gene using lentiviral vectors in MPTP-lesioned rhesus monkeys and in aged rhesus monkeys achieved long-term gene expression and significant functional benefits (Kordower et al., 2000). Kordower and colleagues administered lentiviral-GDNF to the striatum and substantia nigra of nonlesioned aged monkeys and MPTP-treated young monkeys and found extensive expression of GDNF in all of the brains. Lentiviral-delivered GDNF reversed motor deficits in the aged monkeys, and prevented nigrostriatal degeneration and the development of functional deficits in the MPTP-lesioned animals. Another study used a lentiviral vector to achieve long-term delivery of GDNF to the striatum and substantia nigra of aged rhesus monkeys, and found that this treatment conferred significant protective effects on the functioning and integrity of the nigrostriatal pathway (Emborg et al., 2009). Lentiviral delivery of GDNF was also found to increase the number of intrinsic dopaminergic neurones in the primate striatum (Palfi et al., 2002). Dowd et al found that lentiviral-mediated GDNF delivery into the striatum and above the substantia nigra rescued complex motor behaviour (such as corridor, staircase, stepping and cylinder tasks), as well as drug-induced rotational asymmetry, in rats with 6-OHDA lesions of the MFB (Dowd et al., 2005). Lentiviral-mediated delivery is very effective, but there are concerns about its safety, which will have to be addressed before clinical application of this system is feasible.
Thus, gene therapy has shown great promise as a means of achieving long-term and targeted delivery of GDNF to the nigrostriatal system (for reviews see Bjorklund & Lindvall, 2000; Kordower, 2003; Manfredsson et al., 2009). Viral vector technology has also been used to deliver GDNF to the brain in models of other diseases. For example, significant neuroprotective effects were reported in a study that used adenoviral vectors to deliver GDNF to the cortex in an adult rat model of focal cortical trauma (Hermann et al., 2001). Adenoviral-mediated GDNF expression has been reported to rescue rat retinal ganglion cells after axotomy (Schmeer et al., 2002), to protect motoneurones in a transgenic mouse model of amyotrophic lateral sclerosis (Wang, et al., 2002) and to promote functional recovery in a rat model of spinal cord injury (Tai et al., 2003). AAV-2-mediated delivery of GDNF was found to confer neuroprotective effects and motor improvements in both a toxin-mediated adult rat model (McBride et al., 2003) and in a transgenic mouse model of Huntington’s disease (McBride et al., 2006).
Another avenue of exploration is the co-administration of neurotrophic factors with neuronal transplants in cell replacement therapy approaches to PD. Transplantation of embryonic midbrain tissue is a promising and successful therapy for PD, but is limited by the poor survival of the transplanted dopaminergic neurones (for recent reviews, see Brundin et al., 2010; Hedlund & Perlmann, 2009; Olanow et al., 2009). GDNF has been shown by several groups to improve the survival and integration of grafted embryonic dopaminergic neurones in animal models of PD. Rosenblad and colleagues reported that repeated injections of this factor adjacent to embryonic rat ventral midbrain grafts in the 6-OHDA-lesioned rat striatum improved the survival of the grafted dopaminergic cells and induced earlier recovery of motor function than untreated grafts (Rosenblad et al., 1996). Improvements in the survival of grafted dopaminergic neurones and their integration into the host striatum were also reported after pre-incubation of the grafts with GDNF (Apostolides et al., 1998; Granholm et al., 1997; Sullivan et al., 1998; Yurek, 1998). Injection of GDNF along the nigrostriatal tract stimulated the outgrowth of dopaminergic fibres from intranigral grafts towards the striatum (Sinclair et al., 1996; Tang et al., 1998; Wang, Y. et al., 1996). Enhancement of complex motor functions, as well as improved graft survival, were found in 6-OHDA-lesioned rats that had received GDNF-pretreated grafts (Mehta et al., 1998). GDNF pretreatment has also been used to promote the survival of human fetal midbrain tissue, prior to grafting into two PD patients (Mendez et al., 2000). These patients displayed a large increase in fluorodopa uptake after one year, an index of striatal dopaminergic transmission, as measured by positron emission tomography (PET).
Ex vivo gene therapy approaches have been applied in attempts to extend the effects of exogenous GDNF, which is rapidly metabolised in vivo. Genetically-modified embryonic rat midbrain cells which overexpress GDNF have been found to induce earlier functional recovery in 6-OHDA-lesioned rats than control grafts (Bauer et al., 2000). GDNF-overexpressing rat neural precursor cells also significantly increased the survival of co-grafted embryonic dopaminergic neurones (Ostenfeld et al., 2002). Human neural progenitor cells have been used to deliver GDNF, which conferred protective effects on the lesioned nigrostriatal pathway in adult rats (Behrstock et al., 2006). This cellular delivery system, which allows the release of GDNF under an inducible promoter, has also been found to provide GDNF to the aged monkey brain for at least three months (Behrstock et al., 2006). Encapsulation technology involves enclosing cells within a semi-permeable membrane composed of polymer fibres, which allows outward diffusion of any proteins secreted by the cells, while preventing the cells from proliferating extensively and forming tumours. Intrastriatal grafting of an encapsulated GDNF-expressing human (BHK) cell line has been shown to confer neuroprotective and restorative effects in 6-OHDA-lesioned rats (Date et al., 2001; Shingo et al., 2002), particularly when the GDNF-expressing cells are implanted at an early stage of the disease progression (Yasuhara et al., 2005). Encapsulated human fibroblasts genetically engineered to overexpress GDNF were found to exert regenerative effects when implanted into the rat striatum one week after an intrastriatal 6-OHDA lesion (Sajadi et al., 2006). Although the cells were removed after six weeks, the regenerative effects on motor function and on nigral dopaminergic neurones were evident for a further seven weeks, indicating that transient delivery of GDNF was sufficient to confer sustained effects. In MPTP-treated primates, encapsulated GDNF-expressing cells induced transient motor improvements and increases in striatal dopamine uptake, without any adverse side-effects (Kishima et al., 2004). Encapsulated cells expressing GDNF have also been applied in combination with embryonic rat brain grafts in 6-OHDA-lesioned rats, and were found to improve the survival of the grafted dopaminergic neurones and their functional effects (Sautter et al., 1998). Encapsulated cell technology may have great potential for future clinical studies, should the promising effects found in these animal studies be extended to show long-term and safe delivery of appropriate doses of neurotrophic proteins (for reviews see Bensadoun et al., 2003; Lindvall & Wahlberg, 2008).
3.2. Effects of neurturin in vivo
Neurturin has been found to exert protective and functional effects on dopaminergic nigrostriatal neurones after 6-OHDA lesions of the adult rat MFB (Akerud et al., 1999; Horger et al., 1998) or of the striatum (Oiwa et al., 2002; Rosenblad et al., 1999), and after axotomy of the MFB in adult rats (Tseng et al., 1998). The study by Rosenblad and colleagues directly compared the effects of neurturin with those of GDNF in the striatal 6-OHDA lesion model. They found that neurturin was less efficacious than GDNF after intrastriatal and especially after intraventricular delivery, which may reflect poor solubility of neurturin in vivo (Rosenblad et al., 1999). Another study showed that delivery of neurturin into the cerebral ventricles of adult rats using mini-pumps resulted in an increase in striatal dopamine levels (Hoane et al., 1999). A recent study reported that intranigral injection of recombinant neurturin induced increases in striatal dopamine release, which were similar in magnitude to those induced by intranigral injection of recombinant GDNF (Cass & Peters, 2010). Intracerebral delivery of recombinant neurturin has also been found to protect nigrostriatal dopaminergic neurones and induce improvements in motor function in MPTP-treated monkeys (Grondin et al., 2008; Li et al., 2003). Co-administration with recombinant neurturin protein has been reported to improve the survival of fetal rat dopaminergic neurones after intrastriatal grafting into 6-OHDA-lesioned adult rats (Rosenblad et al., 1999).
The need for sustained delivery of neurotrophic proteins to the brain led to studies using viral vector-based delivery systems, which have shown promising results in animal models of PD. Lentiviral gene delivery to the striatum of 6-OHDA-lesioned adult rats of a modified neurturin construct, which had the pro-region deleted and replaced with an immunoglobulin heavy-chain signal peptide, had protective effects on the nigrostriatal pathway (Fjord-Larsen et al., 2005). Sustained functional recovery, with minimal side-effects, was achieved following stereotaxic injection of an adeno-associated virus (AAV) 2–based vector encoding the human neurturin gene, in MPTP-treated monkeys (Kordower et al., 2006) and 6-OHDA-lesioned rats (Gasmi et al., 2007). Stable expression of neurturin using this AAV2 delivery system was achieved for at least a year in rats (Gasmi et al., 2007).
Kordower and colleagues showed that, ten months after treatment with AAV2-neurturin into the striatum and substantia nigra, MPTP-treated parkinsonian monkeys showed a large reduction in the intensity of their motor symptoms compared to buffer-injected animals, which displayed stable symptoms throughout the study. This functional recovery was accompanied by significant preservation of dopaminergic neurones (Kordower et al., 2006). A study using the same expression system in 6-OHDA-lesioned adult rats found long-term neurturin expression in the striatum and dose-dependant protective effects on the nigrostriatal dopaminergic neurones for at least ten months (Gasmi et al., 2007). Similar results were found after application of this system in aged monkeys and no adverse effects were recorded in this study after thorough toxicological testing (Herzog et al., 2007). A further study by this group found that the expression of neurturin using this system could be sustained for a year in rhesus monkeys, as could its therapeutic effects (Herzog et al., 2009). Unlike the case with GDNF, no antibodies to neurturin or no pathological abnormalities were detected after AAV2-delivery in primates (see Bartus et al., 2007).
Neurturin has also been reported to have protective effects on striatal projection neurones in a rat model of Huntington’s disease (Perez-Navarro et al., 2000). AAV-2-mediated delivery of neurturin had neuroprotective effects and induced motor improvements in a genetic mouse model of Huntington’s disease (Ramaswamy et al., 2009).