GDNF expression in the 6-OHDA rat model

The ability of GDNF to protect [34] or restore [126] a lesion of the dopaminergic system in the striatum due to a 6-OHDA application have been demonstrated. Therefore, after having characterized the pSwitch system using EGFP as expressed gene, our goal was to assess if a short pulse of expression from a regulatable vector expressing GDNF instead of EGFP might have any restorative effect on the same 6-OHDA rat model of PD; moreover it was important to verify if the low but detectable expression in the off-state might have a biological effect on animals. With this purpose, animals were first injected with 6-OHDA to induce a DA neuron loss and once the lesion was confirmed by the three motor tests, Apomorphine induced rotation, cylinder test and corridor test, they were divided in 4 groups:

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a) Treated group with the pSwitch system induced with MF b) Not treated group with the pSwitch system not induced c) Positive control expressing GDNF constitutively

d) Negative control expressing EGFP

The first part of the experiment, consisting in inducing the DA neuron lesion in the left striatum, gave the expected results confirmed by all tests: a contralateral rotation of about 250 turns/hour upon apomorphine injection, a preferential left paw use in the cylinder test, a preferential left food withdrawn in the corridor test and a depletion in the total dopamine amount of about 50%

comparing to the unlesioned contra-lateral striatum. Functionality of the viruses was confirmed by GDNF ELISA on animals sacrificed immediately after the first MF induction in the treated group.

Results of this test show that, a high level of expression was given by the GDNF constitutive-expressing virus in the positive control (> 2000 pg/mg tissue), no increase in the NF level was observed in the EGFP negative control (3-4 pg/mg tissue), a two folds increase in GDNF level comparing to the contra-lateral side was measured in animals injected with the pSwitch system but not induced with MF (~6 pg/mg tissue), and a 18 fold overexpression versus the endogenous level when animals injected with the pSwitch system were induced with 20 mg/kg of MF (~54 pg/mg tissue).

The second part of the experiment, which aimed to restore the confirmed lesion by a short MF-induced GDNF expression, leaded to discordant results. In all three motor behavior tests a comparable amelioration was observed but each group presented high standard deviations indicating great variability within the groups. Moreover, in several cases, the motor behavior tests displayed variability in the same animal when tested at the different time points. Dopamine evaluation at the end of the experiment showed that total dopamine level in the striatum was not increased in any of the groups. ELISA for GDNF gave the expected results with a high level of GDNF expression in the positive control, and the basal two-fold overexpression level comparing with the endogenous expression in both groups injected with the pSwitch system; in this case, the pSwitch group induced with MF, was sacrificed four weeks after the last induction and therefore this low level of expression was expected. A TH staining to mark dopaminergic fibers in the striatum was performed but a clear explanation could not be defined. Indeed, in some cases, brains belonging to different rats displayed a similar size of the lesion but the animals were performing completely differently in the motor tests.

Different causes might have leaded to these results: it has been demonstrated that GDNF concentration in cell culture should lie within a specific range otherwise, if a certain threshold is reached, the presence of the protein becomes toxic for cells [127]. In our positive control we measured a concentration of more than 2000 pg/mg tissue, meaning 500-1000 times higher than the normal endogenous level. This high concentration might have lead to invalid results in the positive control.

Another explanation for the variability measured in this experiment can be that the size of the lesion was “border line” meaning that a spontaneous recovery was possibly masking the effect of the treatment. A comparable outcome have been already noticed in a similar experiments in which, after a 6-OHDA lesion, rats were injected with AAV vectors expressing GDNF under the control of the regulatable system Tet-on [57].

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In conclusion more analysis should be accomplished in which the behavioral data, the biochemical data and the results of the dopaminergic neuron staining (TH), are compared at the level of a single animal in order to define a possible pattern in the results or to identify specific loci of the lesion associated with specific motor behavior results.

5.3 Dopamine producing neurons

In PD several areas of the brain display progressive neuronal loss and formation of Lewy bodies and, out of them, the loss of DA neurons in the substantia nigra seems to be responsible for the motor deficits. Therefore it would be of great interest to study the effect of α-synuclein, or the role of other protein involved in PD (LRRK2, DJ-1, Parkin, PINK-1, ATP13A2), directly in this particular population of neurons. Moreover, un-published data showed that, in a free-cell system in vitro, the presence of dopamine can trigger the α-synuclein aggregation hypothesizing a role of the catecholamine in Lewy bodies formation. As DA neurons account only for a small fraction of the total number of neurons in the rat brain, it is not possible to obtain primary culture with more than 10% of DA neurons [110].

LHUMES (Lund human mesencephalic) cells [128] have been used to study the release of dopamine but, being an immortalized cell line, they differ substantially from DA neurons. In alternative, dopaminergic-like neurons can be obtained from the emerging field of stem cells research by starting from different source of pluripotent cells which can be cultured and provided with the appropriate stimuli [127, 129].

In this project we aimed to obtain dopamine-producing neurons by transducing primary cortical neurons with AAVs expressing the essential genes responsible for the catecholamine production.

These genes are TH, to convert L-tyrosine into L-dopa, GCH1, for the synthesis of the TH co-factor BH4, AADC, to convert L-dopa into dopamine, and VMAT-2 which sequestrate dopamine into vesicles. Similar approaches have been already followed by transducing fibroblast with AADC + VMAT-2 and promoting dopamine production by incubating cells with L-dopa [130] or by transducing primary cortical neurons with a tri-cistronic lentiviral vector expressing TH, AADC and GCH-1 and promoting dopamine production by incubating cells with the precursor L-tyrosine.

A high and reproducible dopamine synthesis was obtained by transducing neurons with AADC or with AADC + VMAT and promoting the dopamine production by incubating cells with the precursor L-dopa.

Our experiments agree with those previously published [130] and highlight the importance of the presence of VMAT-2 in increasing both the intra and the extracellular fraction of newly synthesized dopamine. Studies on the amount of the inducer L-dopa, time of incubation, and incubation buffers displayed a good reproducibility of the system regarding both the intra and the extracellular fraction of the synthesized dopamine and its metabolites.

Considering the possible interactions between dopamine and α-synuclein, we showed a lack of toxic effects due to dopamine, α-syn, or the combination of the two, in the neuronal culture at least for the time-frame considered in this experiment (48h). The α-synuclein aggregation tendency in presence of dopamine, observed in a free-cell system in vitro, was not confirmed when western blots were performed on cultured neurons indicating the ability of cells to prevent this aggregation at least for the time considered (96h). Considering the dopamine production, a one tail t-Test showed a tendency of α-synuclein to affect negatively the catecholamine synthesis and release; more

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experiments including extended time points and different amount of synthesized dopamine are needed to confirm this hypothesis.

In conclusion we developed an easy, regulatable and reproducible system for dopamine synthesis in primary cortical neurons that can be used for study the interaction of the neurotransmitter, and its oxidative role, with other components of PD.