Adenovirus-mediated transgene expression in nonhuman primate brain.

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

Gene transfer for neuroprotection in animal models of Parkinson's disease and amyotrophic lateral sclerosis.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for motoneurons (MN) and dopaminergic (DA) neurons, neurons which selectively die in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). GDNF gene delivery has been studied in rodent models of ALS and PD. In a mouse model of ALS, implantation of myoblasts retrovirally transduced with GDNF into hindlimb muscles at 6 weeks of age, i.e. prior to the onset of disease symptoms, increased the number of large MNs that maintained projections to treated muscles at 18 weeks of age. GDNF-treated mice also performed better on tests of motor function and had a delayed onset of disease. In a progressive degeneration rat model of PD, effects of in vivo GDNF gene therapy using an adenoviral vector (AdGDNF) were studied in young and aged rats. AdGDNF protected DA neurons against the neurotoxin, 6-hydroxydopamine (6-OHDA), and was effective whether injected either before or after 6-OHDA damage had commenced. However, if AdGDNF was injected prior to 6-OHDA, it was most effective in protecting against DA-dependent changes in the brain when injected near the terminals of the DA neurons. In contrast, if 6-OHDA damage had already commenced, AdGDNF was most effective if injected near the DA soma. These studies suggest that GDNF gene delivery into specific sites in the CNS or into muscle where MNs have access to secreted GDNF may slow the progression of PD and ALS, respectively. Neurotrophic factor gene therapy offers novel interventions not only for PD and ALS, but also other neurodegenerative diseases and injuries to the nervous system.

Ethanol reduces metabolic uncoupling following experimental head injury.

Previous investigations have shown that ethanol is neuroprotective following experimental traumatic brain injury (TBI). This study sought to determine if the neuroprotective effects of ethanol in a controlled cortical impact (CCI) injury model are related to its effects on cerebral glucose metabolism and blood flow. Adult rats were given ethanol (1.0 g/kg) or saline by intraperitoneal injection followed 40 min later by injury. Regional cerebral blood flow (CBF) and cerebral metabolic rates of glucose (CMRglc) were determined immediately, and at 3, 6, 12, 24, and 72 h postinjury using quantitative autoradiography. Immediately after injury, CMRglc in the contusion core and penumbra was reduced in the ethanol group compared to the saline group: (core CMRglc: 52.2 +/- 16.0 versus 94.2 +/- 14.1 micromol/100 g/min, respectively,p < 0.001; penumbral CMRglc: 58.2 +/- 12.8 versus 82.8 +/-19.7 micromol/100 g/min, respectively; p < 0.05) However, at 24 and 72 h postinjury, penumbral CMRglc in the ethanol group was increased compared to the saline group (p < 0.05 and p < 0.001, respectively). Regarding CBF, contusion core values in the ethanol group were elevated compared to the saline group immediately postinjury, (70.4 +/- 17.1 versus 31.5 +/- 27.8 mL/100 g/min, respectively (p < .05), and at 6, 12, and 24 h postinjury (p < 0.05). Penumbral CBF was also higher at 6 and 72 h in the ethanol group compared to the saline group (p < 0.05). These results indicate that low-dose ethanol is associated with a marked attenuation of immediate postinjury hyperglycolysis and with more normal glucose metabolism in the injury penumbra over the ensuing 3 days. Simultaneously, the reduction in CBF typically seen within the contusion core and penumbra after CCI is less severe when ethanol is present. The net effect of these changes is a decreased degree of uncoupling between glucose metabolism and CBF that otherwise occurs in the absence of ethanol. These changes may likely explain the neuroprotective effect of ethanol.

Pruning of dendrites and restoration of function after brain damage: Role of the NMDA receptor.

Following unilateral injury to the forelimb-representation area of the sensorimotor cortex (FL-SMC) in adult rats, there occurs a biphasic process of overgrowth and partial elimination of neuronal dendrites in layer V pyramidal cells of the homotopic cortex of the opposite hemisphere. These neural events are associated with hyper-reliance on the non-impaired forelimb for postural-supporting and related movements that compensate for impaired function in the other forelimb. The overgrowth appears to be use-dependent because it can be prevented by one-sleeve casts that restrict the range of movements of the unimpaired limb during the period of expected neural growth. In development, "exuberant" growth of neurons is often followed by pruning, a process that has been associated with activity-dependency and a glutamatergic N-methyl-D-aspartate (NMDA) mechanism. To determine whether a related mechanism might be operating in adult animals recovering from brain damage, MK-801, a non-competitive NMDA receptor antagonist, was administered during the pruning phase in adult rats that had sustained FL-SMC lesions. MK-801 prevented the elimination of dendrites in the FL-SMC rats and had no effect on dendritic arborization in Sham-operated rats. MK-801 reinstated dysfunction in the previously-recovered forelimb in FL-SMC rats, and had no effect in Sham-operated rats. These data are consistent with the possibility that there may be a functionally important pruning mechanism with a glutamatergic component in adults with FL-SMC lesions, just as in the developing brain.

Relationship between dendritic pruning and behavioral recovery following sensorimotor cortex lesions.

A unilateral injury to the forelimb area of the sensorimotor cortex results in an increase in dendritic arborization in the contralateral homotopic cortex which is followed by a pruning back of these dendritic arbors. The increase in arborization is due to an increase in the use of the unimpaired forelimb for postural-motor support; whereas, the dendritic pruning is related, in time, to the return to more symmetrical limb use, but is not prevented by the maintenance of asymmetrical limb use. Dendritic pruning can be prevented by administering an NMDA receptor antagonist (such as MK801 or ethanol) during the pruning phase. This manipulation also coincides with the chronic reinstatement of behavioral deficits. The purpose of this study was to see whether removing the antagonism of the NMDA receptor results in the eventual return of dendritic pruning and behavioral recovery. Therefore, MK801 was administered to lesioned animals starting at post-lesion day 18. One group received MK801 injections until day 60 (Lesion + MK60) and another lesioned group received MK801 until day 30 after which the injections were changed to saline until day 60 (Lesion + MK30). Lesion + MK60 animals showed a prevention of dendritic pruning as well as a chronic reinstatement of forelimb deficits. Lesion + MK30 animals also showed a prevention of dendritic pruning, however, they showed behavioral recovery. These findings suggest that pruning of dendritic arbors may not be directly related to behavioral recovery.

Ethanol consumption following recovery from unilateral damage to the forelimb area of the sensorimotor cortex: reinstatement of deficits and prevention of dendritic pruning.

Unilateral injury to the forelimb-representation area of the sensorimotor cortex (FL-SMC) in adult rats results in use-dependent proliferation of dendritic processes, followed by partial pruning, of layer V pyramidal neurons of the contralateral homotopic cortex. In development, 'exuberant' growth of neurons is often followed by pruning, a process that has been associated with a glutamatergic-NMDA receptor mechanism. A related mechanism may play a role in injury-related pruning of dendrites in adults. The N-methyl-D-aspartate (NMDA) receptor antagonist MK801, administered throughout the pruning phase to adult animals with FL-SMC lesions, prevents dendritic pruning and disrupts behavioral recovery. Ethanol (ETOH) also acts as an NMDA receptor antagonist. It has been shown to reduce NMDA-active ion currents, inhibit NMDA-evoked electrophysiological responses, and decrease glutamate-binding in the hippocampus and cortex. ETOH also affects neuromorphology in the developing and adult cerebellum, hippocampus, and cortex. Ethanol's involvement with NMDA receptor function and its influence on dendritic morphology led us to examine its effect on dendritic pruning and behavioral recovery following unilateral FL-SMC lesions. Lesioned animals were exposed to moderate doses of ethanol in a liquid diet only during the period of dendritic pruning. As with MK801, ETOH prevented pruning and reinstated chronic behavioral asymmetries.

Use-dependent exacerbation of brain damage occurs during an early post-lesion vulnerable period.

For a period of time after unilateral brain injury, surviving neural tissue surrounding the lesion may be vulnerable to extremely high behavioral demand. Previously, we found that lesions of the forelimb representation area of the sensorimotor cortex (FL-SMC) in rats increase in size substantially when the intact forelimb is immobilized with a plaster of paris cast during the first 15 days after surgery, which forces overuse of the impaired forelimb. The present study was designed to determine whether the adult brain is more vulnerable to forced overuse of the impaired forelimb during the first 7 days post-lesion than during the second 7 days post-lesion. Using behavioral tests of forelimb use and stereological analysis of remaining tissue volume 40 days after FL-SMC lesions, we found that forced overuse of the impaired forelimb during the first 7 days after the initial damage caused expansion of neural injury and greatly interfered with restoration of function. In contrast, forced overuse of the impaired forelimb during the second 7 days had no significant effect on lesion size but nevertheless interfered with restoration of function. Thus, surviving neural tissue in the damaged hemisphere and recovery of function appear to be vulnerable to prolonged forced overuse of the impaired forelimb throughout the first 15 days, but tissue loss was detectable only when the animal was forced to use the impaired forelimb during the first 7 days after injury.

Quantitative analyses of GFRalpha-1 and GFRalpha-2 mRNAs and tyrosine hydroxylase protein in the nigrostriatal system reveal bilateral compensatory changes following unilateral 6-OHDA lesions in the rat.

Copy numbers of mRNAs for GFRalpha-1 and GFRalpha-2, the preferred receptors for glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) were determined by real-time quantitative RT-PCR (QRT-PCR). Receptor expression was assessed in striatum (ST) and substantia nigra (SN) of normal rats and rats acutely or progressively lesioned by 6-OHDA injected into the medial forebrain bundle or ST, respectively. GFRalpha-1 mRNA was clearly detected in normal ST. In normal SN, significantly higher expression of both receptors was observed. At 4 weeks after acute lesion, GFRalpha-2 mRNA was markedly decreased in SN bilaterally, whereas GFRalpha-1 mRNA in SN and ST was not affected. A progressive lesion resulted in a progressive decrease of GFRalpha1 mRNA in ST bilaterally. In SN, levels of GFRalpha-1 mRNA were not significantly affected by a progressive lesion, whereas GFRalpha-2 mRNA was markedly decreased bilaterally. Quantitative western blotting standardized against tyrosine hydroxylase (TH) protein from PC12 cells revealed the expected decrease in TH protein in lesioned SN, but also significant increases in TH protein in contralateral, unlesioned SNs at 4 weeks after both acute and progressive lesions. These data suggest that previously unrecognized compensatory changes in the nigrostriatal system occur in response to unilateral dopamine depletion. Since the changes observed in receptor expression did not always parallel loss of dopamine neurons, cells in addition to the nigral dopamine neurons appear to be affected by a 6-OHDA insult and are potential targets for the neurotrophic factors, GDNF and NTN.

Glial cell line-derived neurotrophic factor (GDNF) as a defensive molecule for neurodegenerative disease: a tribute to the studies of antonia vernadakis on neuronal-glial interactions.

Research stemming from interests in neuronal-glial interactions has led to the identification of a number of novel trophic factors, such as the dopaminergic neurotrophic factor glial cell line-derived neurotrophic factor (GDNF). Delivery of the GDNF gene to rat models of Parkinson's disease suggests a potential clinical use of GDNF gene therapy for humans with this disease. This review article briefly summarizes the history of GDNF and the effects of GDNF gene delivery prior to or after a lesion of the rat nigrostriatal system.

Use-dependent structural events in recovery of function.

We described research suggesting that forelimb use is essential for marked neural growth in the intact cortex after unilateral forelimb-cortical lesions. Although unilateral brain injury can cause severe functional impairment, the injury may be capable of mobilizing potent resources for compensatory changes such as dendritic arborization in the noninjured hemisphere, but only for a limited period of time and only with appropriate behavioral pressure. Unexpectedly, surviving tissue in the injured hemisphere may be fatally vulnerable to excessive behavioral demand. If the impaired limb is overused because the nonimpaired limb is restricted by a one-sleeve cast, injury size is greatly increased and recovery of function is severely disrupted. It is hypothesized that behaviorally driven neurotransmitter release relating to forced use of the forelimb may be toxic to surviving tissue that has been partially traumatized by the lesion. These data and hypotheses are summarized in Fig. 7. The "use-it-or-lose-it" rehabilitative approach is popular, but perhaps a less aggressive strategy should be adopted for optimal restoration of function in the injured hemisphere. Whereas traditional experiments on mechanisms of recovery of function are designed specifically to determine potentially compensatory neural changes that might mediate behavioral outcome, these experiments support a quite different view of the interplay between neural and behavioral events: behavioral changes may directly alter anatomical events.

Combining glial cell line-derived neurotrophic factor gene delivery (AdGDNF) with L-arginine decreases contusion size but not behavioral deficits after traumatic brain injury.

Our laboratory has previously demonstrated that viral administration of glial cell line-derived neurotrophic factor (AdGDNF), one week prior to a controlled cortical impact (CCI) over the forelimb sensorimotor cortex of the rat (FL-SMC) is neuroprotective, but does not significantly enhance recovery of sensorimotor function. One possible explanation for this discrepancy is that although protected, neurons may not have been functional due to enduring metabolic deficiencies. Additionally, metabolic events following TBI may interfere with expression of therapeutic proteins administered to the injured brain via gene therapy. The current study focused on enhancing the metabolic function of the brain by increasing cerebral blood flow (CBF) with l-arginine in conjunction with administration of AdGDNF immediately following CCI. An adenoviral vector harboring human GDNF was injected unilaterally into FL-SMC of the rat immediately following a unilateral CCI over the FL-SMC. Within 30min of the CCI and AdGDNF injections, some animals were injected with l-arginine (i.v.). Tests of forelimb function and asymmetry were administered for 4weeks post-injury. Animals were sacrificed and contusion size and GDNF protein expression measured. This study demonstrated that rats treated with AdGDNF and l-arginine post-CCI had a significantly smaller contusion than injured rats who did not receive any treatment, or injured rats treated with either AdGDNF or l-arginine alone. Nevertheless, no amelioration of behavioral deficits was seen. These findings suggest that AdGDNF alone following a CCI was not therapeutic and although combining it with l-arginine decreased contusion size, it did not enhance behavioral recovery.

Effect of AdGDNF on dopaminergic neurotransmission in the striatum of 6-OHDA-treated rats.

We have previously observed that the delivery of an adenoviral vector encoding for glial cell line-derived neurotrophic factor (AdGDNF) into the substantia nigra (SN) 7 days after intrastriatal administration of 6-hydroxydopamine (6-OHDA) protects dopamine (DA)-dependent behaviors, tyrosine hydroxylase immunoreactive (TH+) cells in SN, and amphetamine-induced c-fos induction in striatum. In the present study, we sought to determine if the behavioral protection observed in 6-OHDA-treated rats receiving AdGDNF was associated with an increase in DA availability in the striatum as measured by microdialysis. Rats received intrastriatal 6-OHDA (16 microg/2.8 microl) or vehicle followed 7 days later by intranigral AdGDNF (3.2x10(7) pfu/2 microl), AdLacZ (3.2 x 10(7) pfu/2 microl), or phosphate buffered saline (PBS). Three weeks later, microdialysis samples were collected from the same striatal region under basal conditions, following KCl (100 mM) or amphetamine (250 microM) administered via the striatal microdialysis probe, or amphetamine administered systemically (6.8 mg/kg i.p). Animals given 6-OHDA followed by either PBS or AdLacZ showed a decrease in basal extracellular striatal DA levels to 24% of control. In contrast, basal extracellular DA in 6-OHDA-lesioned rats with a nigral injection of AdGDNF was almost 3-fold higher than 6-OHDA-vehicle treated animals, 65% of control DA levels. Moreover, although KCl and amphetamine produced no increase in striatal DA release in 6-OHDA-treated rats that subsequently were given either PBS or AdLacZ, these manipulations increased DA levels significantly in 6-OHDA-treated rats later given AdGDNF. Thus, DA neurotransmission within the striatum of 6-OHDA treated rats appears to be enhanced by increased expression of GDNF in the nigra.

Use-dependent exaggeration of neuronal injury after unilateral sensorimotor cortex lesions.

Unilateral injury to the forelimb representation area of the sensorimotor cortex (FL-SMC) in adult rats causes over-reliance on the unimpaired forelimb for postural-motor movements, as well as overgrowth of layer V pyramidal cell dendrites in the homotopic cortex of the noninjured hemisphere. The overgrowth appears to be use-dependent because it can be prevented by restricting movements of the unimpaired forelimb. Additionally, restricting the unimpaired forelimb in animals with FL-SMC damage results in significantly greater behavioral dysfunction when examined 2 d after cast removal (compared to that after impaired-limb immobilization, or no limb immobilization). In the present study, the long-term behavioral and anatomical effects of limb immobilization were examined. Animals with FL-SMC lesions were fitted with casts immediately after the lesion that immobilized the impaired forelimb, the unimpaired forelimb, or neither forelimb for 15 d. Immobilization of the nonimpaired forelimb resulted in chronic prevention of dendritic growth and severe and chronic behavioral deficits. In addition, immobilization of the nonimpaired forelimb resulted in a dramatic exaggeration of the neuronal injury, presumably attributable to forced overuse of the impaired limb. Immobilization of the impaired forelimb resulted in no detectable neural changes and in only slightly increased and longer-lasting behavioral asymmetries (compared to nonimmobilized, lesioned animals), presumably attributable to mild disuse of the impaired limb. Immobilization of a single forelimb in nonlesioned rats resulted in no significant behavioral or anatomical changes. Together, these results suggest that although behavioral experience can enhance neural growth after brain injury, the region surrounding the injury may be vulnerable to behavioral pressure during the early postlesion period.

Use-dependent exaggeration of brain injury: is glutamate involved?

Extreme overreliance on the impaired forelimb following unilateral lesions of the forelimb representation area of the rat sensorimotor cortex (FL-SMC) leads to exaggeration of the initial cortical injury. Glutamate has repeatedly been implicated in the secondary processes leading to neuronal death following traumatic insult, chiefly because of the neuroprotective properties of excitatory amino acid antagonists in a variety of animal models of brain injury. The present study investigated the possibility that NMDA receptor-mediated processes are involved in use-dependent exaggeration of neuronal injury. Rats were fitted with one-sleeved casts that immobilized the intact forelimb for the first 7 days following FL-SMC lesion, a procedure previously shown to result in use-dependent exaggeration of injury and more severe and persistent limb-use deficits. In the present investigation, administration of MK-801 (1 mg/kg ip once daily on alternate days) during the casting period spared neural tissue surrounding the lesion and enhanced functional recovery of the impaired forelimb. These results suggest a role for NMDA receptor-mediated processes in use-dependent exaggeration of injury.

Delivery of a GDNF gene into the substantia nigra after a progressive 6-OHDA lesion maintains functional nigrostriatal connections.

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinson's disease through rescuing compromised dopaminergic neurons.

Quantitative analysis of transgene protein, mRNA, and vector DNA following injection of an adenoviral vector harboring glial cell line-derived neurotrophic factor into the primate caudate nucleus.

Gene therapy for neurodegenerative diseases relies on stable expression of a vector-mediated transgene in the human central nervous system (CNS). In nonhuman primate CNS, transgene expression has been primarily assessed using descriptive histological methods. Here, we quantified the expression of a human glial cell line-derived neurotrophic factor (hGDNF) transgene using an ELISA specific for hGDNF protein and real-time quantitative RT-PCR and PCR for hGDNF mRNA and vector DNA, respectively. Transgene expression was assessed 1 week after injection of an E1-, E3-deleted adenovirus harboring hGDNF into the caudate nucleus of St. Kitts green monkey. We found that 57-147 million and 116-771 million copies of hGDNF mRNA and vector DNA, respectively, were present per 10,000 copies of the beta-actin gene. In the same sites, 40-152 pg of hGDNF protein per milligram of tissue was measured. Comparisons of these measures among monkeys demonstrated variable vector DNA and protein levels, but consistent mRNA levels at one-third of the level of vector DNA. This suggests that local responses to the vector play a role in the level of transgene expression and that high levels of vector DNA do not necessarily predict a high level of transgene protein. However, the results of this study do show that neuroprotective levels of GDNF transgene expression can be achieved following injection of an adenoviral vector into nonhuman primate caudate. Moreover, these assays provide quantitative methods for evaluating and comparing viral vectors in primate CNS.

Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged Parkinsonian rat.

Injection of an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic (DA) neurons in the substantia nigra (SN) of young rats. As Parkinson's disease occurs primarily in aged populations, we examined whether chronic biosynthesis of GDNF, achieved by adenovirus-mediated delivery of a GDNF gene (AdGDNF), can protect DA neurons and improve DA-dependent behavioral function in aged (20 months) rats with progressive 6-OHDA lesions of the nigrostriatal projection. Furthermore, the differential effects of injecting AdGDNF either near DA cell bodies in the SN or at DA terminals in the striatum were compared. AdGDNF or control vector was injected unilaterally into either the striatum or SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the same side as the vector injection. AdGDNF injection into either the striatum or SN significantly reduced the loss of FG labelled DA neurons 5 weeks after lesion (P

Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration.

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for beta-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [(125)I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 alpha-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Inflammatory responses and their impact on beta-galactosidase transgene expression following adenovirus vector delivery to the primate caudate nucleus.

An E1, E3 deleted adenovirus vector, serotype 5, carrying the marker gene LacZ was bilaterally microinfused into the caudate nuclei of 10 St Kitts green monkeys. The location and number of cells expressing transgene and host immunologic response were evaluated at 1 week (n = 2) and 1 month (n = 8) following vector infusion. A large number of cells expressed beta-galactosidase in some monkeys, exceeding 600000 in one monkey, but no expression was seen in three of 10. All monkeys had positive adenoviral antibody titers before vector infusion, indicating the possibility of previous exposure to some adenovirus, but only one showed a significant increase in titer afterwards. Inflammatory cell markers revealed an inverse correlation between transgene expression and the extent of inflammatory response. Dexamethasone administered immediately before and for 8 days following vector delivery, however, had no effect on transgene expression. The demonstration of significant inflammatory responses in the brain of some individual primates, including demyelination, indicates the need for new generations of adenovirus vectors, or the successful suppression of inflammatory responses, before this vector is suitable for non-cytotoxic clinical applications in the CNS.