Introduction

Disorders with Lewy body (LB) formation such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are leading causes of cognitive and motor dysfunction in the aging population. However, only a few treatment methods are currently available. In recent years, considerable progress has been made in developing new therapies for these neurodegenerative disorders utilizing gene transfer strategies.¹ Since nigral dopaminergic loss is a common substrate for the neurological deficits in patients with these disorders,² most gene therapy approaches have been directed at protecting or rescuing neurons of the nigral–striatal (NS) pathway with vectors expressing antiapoptotic factors, neurotrophic agents and enzymes relevant to this circuitry.¹ However, it might be necessary to develop alternative gene therapy approaches in view of increasing evidence that favors a potential role for abnormal aggregation of -synuclein in the central nervous system (CNS) as one of the central mechanisms involved in the pathogenesis of PD and DLB.^{3, 4, 5} -Synuclein is capable of self-aggregating to form both oligomers and polymers.^{6, 7, 8} Toxic oligomers form protofibrils that can potentially damage the cell membrane and promote neurodegeneration.^{9, 10} While in PD -synuclein accumulation occurs primarily in the striatal nigral system, in DLB -synuclein also accumulates in the neocortex and hippocampus.

In familial forms of parkinsonism, mutations accelerate aggregation^{11, 12} and toxic conversion of -synuclein.^{13, 14} In sporadic forms of PD and DLB it is less clear what triggers -synuclein aggregation; however, recent studies suggest that interactions between environmental factors promoting oxidative stress and genetic polymorphisms¹⁵ might trigger a shift in the balance between factors promoting and blocking -synuclein aggregation.¹⁶ Therefore, an alternative therapy for PD and DLB might require the transfer of genes that might block the pathological aggregation of -synuclein.¹ Among them, three potential candidates have been recently identified, namely -synuclein, Parkin and heat-shock proteins (Hsp).^{1, 17}

-Synuclein belongs to a larger family of synaptic proteins that includes -synuclein and -synuclein.¹⁸ -Synuclein is an abundant synaptic protein originally identified as PNP14 in the bovine brain.^{19, 20} The most significant difference between the synucleins is that -synuclein lacks the majority of the hydrophobic nonamyloid component (NAC) domain.²¹ This highly amyloidogenic region is responsible for the self-aggregating capacity of -synuclein.^{21, 22} In contrast to -synuclein, -synuclein does not aggregate under stress conditions or form insoluble oligomers and is capable of blocking -synuclein aggregation both in vivo and in vitro.²¹ At a critical ratio, -synuclein might regulate the state of aggregation of -synuclein; therefore alterations in this molecule might be associated with PD and DLB.²¹ Remarkably, recent studies support this possibility by showing that levels of -synuclein were decreased in brain regions selectively affected in DLB, such as the superior temporal cortex, the cingulate and substantia nigra (SN).²³

Taken together, these studies suggest that in vivo transfer of genes encoding for -synuclein might oppose the deleterious effects of -synuclein and possibly provide a novel strategy in the development of experimental treatments for PD and DLB. To assess this possibility and to better understand the mechanisms involved, a lentiviral vector expressing human (h) -synuclein (lenti--synuclein) was prepared and tested in a transgenic (tg) model of DLB where -synuclein accumulation was abundant in the neocortex and hippocampus. This approach was favored over other viral vectors because lentiviral vectors have been shown to efficiently transduce cells of the CNS and are not cytotoxic.^{24, 25, 26} Furthermore, expression of the therapeutic product can theoretically be sustained for the life span of the animal.

Results

Expression of -synuclein in transduced neuronal cells and the mouse CNS

Rat neuroblastoma B103 cells were infected with lentiviral vectors expressing either -synuclein or green fluorescent protein (GFP) alone (Figure 1a). Cells infected with the lenti--synuclein vector (multiplicity of infection (m.o.i.) 50) produced an activity significantly higher than that of lenti-GFP infected cells. Fluorescent microscopy analysis showed that the lentiviral vector infected over 95% of the cells and that GFP or -synuclein (Figure 1b–d) was efficiently expressed. Western blot (WB) analysis confirmed that neuronal cells transduced with lenti--synuclein expressed high levels of this protein, as opposed to undetectable expression in wild type (wt) cells. The protein was detected as a single band at an approximate molecular weight (MW) of 20 kDa and found only in the cytosolic fraction (Figure 1e). Further analysis was performed in vivo by injecting lenti--synuclein into the brains of nontransgenic (nontg) and h-synuclein tg mice. Consistent with the in vitro studies, immunocytochemical analysis showed that compared to the vector control (Figure 1f), lenti--synuclein was expressed by neurons in the neocortex (Figure 1g) and hippocampus (Figure 1h) within a 250 m radius around the site of the injection. Double immunocytochemical experiments with the neuronal marker NeuN and -synuclein confirmed that greater than 85% of the neurons within this region expressed the lentiviral-driven -synuclein product (data not shown). The distribution of -synuclein expression was similar to the distribution of GFP after injection with lenti-GFP vector (data not shown). The ribonuclease protection assay (RPA) corroborated the presence of -synuclein mRNA in the brain homogenates of mice injected with lenti--synuclein (Figure 1i).

Figure 1.

Lenti--synuclein expression in a neuronal cell line. (a) Vector design showing the lentiviral construct expressing -synuclein. The internal promoters driving the transgenes are indicated by arrows (CMV). All vectors utilized the WPRE. Long terminal repeat (LTR) sequences are shown on the ends (packaging signal, ). B103 neuroblastoma cells were transduced (1.0 ng/well, or 0.015 pg p24 gag-antigen per cell) with lenti-vector control (b), lenti-GFP (c) or lenti--synuclein (d) and imaged 5 days later with the confocal microscope. Immunocytochemical analysis showed that over 90% of the transduced cells expressed -synuclein. (e) Immunoblot analysis of stably transfected h-synuclein B103 neuroblastoma cells that were then transduced with either the lenti-vector control (-), lenti-GFP or lenti--synuclein. The upper image corresponds to the blot probed with the anti--synuclein antibody. The lower image corresponds to the blot probed with the anti--synuclein antibody. Lenti--synuclein product was detected in the cytosolic (cyt.) but not in the particulate (part.) fraction of the h-synuclein transfected B103 neuroblastoma cells. No h- or -synuclein expression was detected in B103 cells that were transduced with the lenti-vector control. (f–h) Lenti--synuclein expression in the murine brain. Nontg mice received a single intracerebral lentiviral injection, and were analyzed 4 weeks later by confocal microscopy and immunocytochemistry with an antibody against -synuclein. Low-power ( 40) view of the neocortex in mice injected with lenti-vector control (f) or lenti--synuclein (g). (h) Low-power view of the hippocampus in a mouse injected with lenti--synuclein. (i) Analysis of h- and -synuclein mRNA expression by RPA in nontg and h-synuclein tg mice transduced with lentiviral vectors. Lane 1 shows undigested riboprobes (U). Abundant -synuclein mRNA was detected in tg mice that received intracerebral lenti--synuclein injection. (b–d) Bar=20 m; (f–h) bar=50 m.

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Lenti--synuclein reduces the neuronal accumulation of h-synuclein and ameliorates the synaptic alterations in tg mice

To assess the potential effects of -synuclein in reducing h-synuclein accumulation in vivo, h-synuclein tg mice that develop inclusion bodies, synaptic loss and dopaminergic deficits²⁷ received an intracerebral injection with a lentiviral vector expressing either -synuclein or GFP. Immunocytochemical analysis demonstrated that h-synuclein tg mice injected with lenti--synuclein showed a considerable reduction in the accumulation of h-synuclein in the neuronal cell bodies and in the formation of inclusion bodies in the neocortex (Figure 2a, b, e, f) and hippocampus (Figure 2i, j), when compared to h-synuclein tg mice treated with lenti-GFP (Figure 2c, d, g, h, k, l) or heat-inactivated lenti--synuclein (data not shown). Transgenic mice injected with lenti--synuclein (Figure 2m) displayed significantly lower levels of h-synuclein positive inclusions ipsilateral to the site of injection in both the neocortex and hippocampus, whereas contralateral to the injection, levels are comparable to lenti-GFP-treated tg mice (Figure 2n). Double immunocytochemical studies (Figure 3a–d) confirmed these observations and showed that compared to vector controls (Figure 3c), transduced neurons expressing high levels of lenti--synuclein displayed reduced accumulation of h-synuclein (Figure 3d). In neurons of the treated mice where both synucleins were coexpressed, h-synuclein was identified as discrete, granular aggregates in the cytoplasm and in the perinuclear region (Figure 3d).

Figure 2.

Lenti--synuclein reduces the neuronal accumulation of h-synuclein in tg mice. h-Synuclein tg mice (12 months old) received intracerebral lentiviral injections into the neocortex and hippocampus, and were analyzed 4 weeks later by immunocytochemistry and bright-field digital microscopy. Panels (a–d) are low power views ( 40) and (e–l) are higher power views ( 400) of the neocortex and hippocampus contralateral (contra) and ipsilateral (ipsi) to the site of injection. (a, e, i) Abundant h-synuclein accumulation in the neuropil and neuronal cell bodies in the contralateral hemibrain. (b, f, j) Lenti--synuclein injection resulted in decreased h-synuclein accumulation in the neuropil and neuronal cell bodies in tg mice. Treatment with lenti-GFP had no effects in the patterns of h-synuclein accumulation ipsilateral (c, g, n) or contralateral (d, h, l) to the injection site. (m, n) Comparison of levels of h-synuclein positive inclusions in the neocortex and hippocampus contra and ipsilateral to the site of injection with lenti--synuclein and lenti-GFP, respectively. ^*Significant difference in sites ipsilateral to the injection site compared to control region (P<0.05, one-way ANOVA posthoc Dunnett's). (a–d) Bar=50 m; (e–l) bar=20 m.

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Figure 3.

Double immunocytochemical analysis of patterns of h- and -synuclein expression in the brains of tg mice treated with lentiviral vectors. All images were obtained by LSCM of the neocortex of h-synuclein tg mice (12 months old). Panels a and b correspond to sections labeled with an antibody against -synuclein. Panels c and d correspond to merged images from sections double labeled with antibodies against -synuclein (red) and -synuclein (green). (a) No reactivity is observed in neurons of mice treated with lenti-vector control. (b) Transduction with lenti--synuclein resulted in high levels of expression of this protein in pyramidal neurons. (c) Abundant h-synuclein accumulation in neuronal cell bodies and synapses of tg mice treated with lenti-GFP. (d) h-Synuclein accumulation in neuronal cell bodies and synapses is reduced in tg mice treated with lenti--synuclein. (e–h) Double immunocytochemical analysis of patterns of human h-synuclein (red) and synaptophysin (green) expression in the brains of mice treated with lentiviral vectors. (e) Nontransgenic mice treated with vector control. (f, g) In tg mice treated with lenti-GFP there is abundant h-synuclein accumulation in neuronal cell bodies and synapses. (g) Merged image of tg mouse treated with lenti-GFP. (h) Merged image of tg mouse treated with lenti--synuclein. (i) Percent of synapses with h-synuclein in nontg and tg mice treated with lenti-GFP and lenti--synuclein. (j) Percent area of neuropil covered, as identified by the synaptic marker synaptophysin in nontg and tg mice treated with lenti-GFP and lenti--synuclein. ^*Significant difference compared to h-synuclein tg mice treated with lenti-GFP (P<0.05, one-way ANOVA post hoc Tukey–Kramer). Bar=10 m.

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Under physiological conditions -synuclein is localized primarily to the presynaptic boutons²⁸ and in LBD and in the tg mice, increased accumulation of h-synuclein in the synapses is associated with functional deficits and synapse loss.²¹ To ascertain the effects of lenti--synuclein treatment on h-synuclein accumulation in the nerve terminals, double immunolabeling studies with antibodies against the presynaptic terminal marker synaptophysin and h-synuclein were performed. Confocal imaging of double-labeled sections showed that in comparison to h-synuclein tg mice injected with lenti-GFP (Figure 3g), those that were injected with lenti--synuclein displayed decreased accumulation of h-synuclein in synaptophysin-immunoreactive nerve terminals in the neocortex (Figure 3h) and hippocampus (data not shown). Consistent with these findings, image analysis showed that while in lenti-GFP or vector control-treated tg mice, approximately 70% of the synapses displayed intense transgene-derived h-synuclein immunoreactivity, in mice treated with lenti--synuclein the proportion was reduced to 25% (Figure 3i). This was accompanied by an amelioration of the neurodegenerative changes, because compared to mice injected with lenti-GFP (Figure 3g, j) or vector alone, h-synuclein tg mice that were injected with lenti--synuclein displayed levels of synaptophysin immunoreactivity in the neocortex (Figure 3h, j) similar to the nontg controls (Figure 3e, f).

Lenti--synuclein reduces membrane translocation of h-synuclein and promotes Akt activation

Since it is possible that abnormal accumulation of -synuclein in synapses and neuronal cell bodies is associated with increased translocation of -synuclein from the soluble to the insoluble fraction, we performed WB analysis with the cytosolic and particulate fractions of tg mice treated with lenti--synuclein. As expected, most of the h-synuclein monomeric form was present in the cytosolic fraction (Figure 4a, c). Compared to the cytosolic fraction, in the particulate fraction there was greater accumulation of h-synuclein oligomers in the brains of the empty vector control-treated h-synuclein tg mice (Figure 4b, c). Treatment with lenti--synuclein resulted in a significant decrease in the levels of all forms of h-synuclein in the particulate fraction (Figure 4b, c). Reduction of h-synuclein oligomers and monomers in the particulate fraction of brain homogenates of tg mice treated with lenti--synuclein corresponds to an increase in monomers in the cytosolic fraction. These studies suggest that -synuclein might interact with -synuclein, preventing its abnormal accumulation and membrane translocation. To investigate this possibility, immunoprecipitation studies were performed. This analysis showed that h-synuclein was coimmunoprecipitated with -synuclein in the brains of tg mice treated with lenti--synuclein but not in mice treated with the lenti-GFP vector control (Figure 4d).

Figure 4.

Western blot analysis of the effects of lenti--synuclein treatment in the brains of tg mice. Brain homogenates from 12-month-old mice were divided into cytosolic (cyt.) and particulate (part.) fractions and probed with an anti--synuclein antibody. (a, b) Monomeric h-synuclein was detected as a band of a molecular weight of 19 kDa while h-synuclein oligomers were detected as bands within a range of 60–100 kDa. Compared to the cytosolic fraction (a), in the particulate fraction (b), there was greater accumulation of h-synuclein oligomers in the brains of the control lenti-GFP-treated h-synuclein tg mice. Compared to lenti-GFP-treated mice (lanes 1–4), treatment with lenti--synuclein resulted in a decrease in the levels of all forms of h-synuclein in the particulate fraction (b, lanes 5–8) and an increase in monomers in the cytosolic fraction (a, lanes 5–8). VersaDoc-aided quantitative analysis of the levels of h-synuclein showed a significant increase in monomers in the cytosolic fraction and a significant decrease in oligomers in the particulate fraction in the lenti--synuclein-treated tg mice. ^*Significant difference compared to h-synuclein tg mice treated with lenti-GFP (P<0.05, one-way ANOVA posthoc Tukey–Kramer). (d) Coimmunoprecipitation of h-synuclein with -synuclein in brain homogenates from lenti--synuclein-treated tg mice. Samples from either lenti--synuclein-treated tg mice (lanes 5–8) or lenti-GFP-treated tg mice (lanes 9–12) were fractionated into the cytosolic (cyt.) fractions (lanes 1, 3, 5, 6, 9, 10) and the particulate (part.) fractions (lanes 2, 4, 7, 8, 11, 12). Each sample (200 g) was immunoprecipitated with either pre-immune serum (lanes 5, 7, 9, 11) or anti--synuclein serum (lanes 6, 8, 10, 12), respectively, followed by immunoblotting with anti--synuclein Syn-1 (upper panel). The filter was reprobed with anti--synuclein antibody (lower panel). 10% input controls are simultaneously shown as positive controls (lanes 1–4). Note that h-synuclein coimmunoprecipitates with -synuclein in the cytosolic fractions (lane 6) and to a lesser extent in the particulate (lane 8) fractions in lenti--synuclein-transduced cells. For all experiments, similar results were obtained in three independent experiments.

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To further verify these findings in an independent model system, B103 neuroblastoma cells transfected with h-synuclein were treated with lenti--synuclein or lenti-GFP. Consistent with previous studies,²¹ compared to vector controls (Figure 5a), cells transfected with h-synuclein displayed widespread accumulation of h-synuclein in the neuronal cell bodies and neuritic processes (Figure 5b). Treatment of h-synuclein-transfected cells with the lenti-GFP (Figure 5c) had no apparent effect; in contrast, treatment of transfected cells with lenti--synuclein resulted in a decreased accumulation of h-synuclein both in the perikaryon and neurites (Figure 5d). In agreement with this observation, WB analysis showed that compared to the vector control, in cells transfected with h-synuclein there was a significant accumulation of oligomers in the particulate fraction and low levels of monomeric h-synuclein (Figure 5e). Treatment with vector alone or lenti-GFP had no effects. In contrast, in cells transduced with the lenti--synuclein there was a significant decrease in h-synuclein oligomers and the appearance of a prominent band corresponding to the monomeric form of h-synuclein (Figure 5e). Furthermore, and in agreement with the studies in tg mice (Figure 4d), immunoprecipitation experiments with the transfected cell lines followed by WB showed that h-synuclein was coimmunoprecipitated with -synuclein (Figure 5f). Taken together, these results are consistent with the known capacity of -synuclein to act as a chaperone molecule²⁹ and suggests that the protective effects of -synuclein might be related to its ability to bind other molecules. Among them, recent in vitro studies have shown that -synuclein binding to Akt stabilizes this molecule, resulting in its activation. This increased activity promotes neurite outgrowth and protects neurons from the neurotoxic effects of h-synuclein.³⁰ Consistent with this possibility, WB analysis of the brains of tg mice treated with the lentiviral vectors showed that compared to lenti-GFP control, in mice treated with lenti--synuclein the levels of phospho (p) Akt, an activated form of this kinase, were higher (Figure 6). Taken together, these results indicate that -synuclein might protect against the neurotoxic effects of -synuclein via activation of Akt signaling.

Figure 5.

Reduced h-synuclein accumulation in neuronal cell line transduced with lenti--synuclein. Panels a–d are from neuroblastoma B103 cells double labeled with antibodies against h-synuclein (green) and -synuclein (red) and imaged with the LSCM. (b) Cells transfected with h-synuclein displayed widespread accumulation of h-synuclein in the neuronal cell bodies and neuritic processes. (c) Treatment of h-synuclein-transfected cells with the lenti-GFP had no apparent effect. (d) Treatment of transfected cells with lenti--synuclein resulted in a decreased accumulation of h-synuclein both in the perikaryon and neurites. (e) Western blot analysis showed that compared to the vector control, in cells transfected with h-synuclein there was a significant accumulation of oligomers in the particulate fraction and low levels of monomeric h-synuclein. Lenti--synuclein treatment of h-synuclein-transfected cells (lane 5) shows a significant decrease in oligomers and a corresponding increase in the monomeric form of h-synuclein. (f) Coimmunoprecipitation of h-synuclein with -synuclein in lenti--synuclein-transduced cells. Extracts from either lenti--synuclein-transduced cells (lanes 5–8) or lenti-GFP-transduced cells (lanes 9–12) were fractionated into the cytosolic (cyt.) fractions (lanes 1, 3, 5, 6, 9, 10) and the particulate (part.) fractions (lanes 2, 4, 7, 8, 11, 12). Each sample (200 g) was immunoprecipitated with either pre-immune serum (lanes 5, 7, 9, 11) or anti--synuclein serum (lanes 6, 8, 10, 12), respectively, followed by immunoblotting with anti--synuclein Syn-1 (upper panel). The filter was reprobed with anti--synuclein antibody (lower panel). 10% input controls are simultaneously shown as positive controls (lanes 1–4). Note that h-synuclein coimmunoprecipitates with -synuclein in the cytosolic fractions (lane 6) and to a lesser extent in the particulate (lane 8) fractions in lenti--synuclein-transduced cells. For all experiments, similar results were obtained in three independent experiments. Bar=10 m.

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Figure 6.

Effects of treatment with lenti--synuclein on pAkt expression in the brains of tg mice. Brain samples from 12-month-old h-synuclein tg mice were homogenized and soluble fractions were extracted and analyzed utilizing antibodies against phospho (p) and total (t) Akt. Immunoblot analysis for pAkt (Ser473) (a, upper band) and tAkt (a, lower band) show increased pAkt immunoreactivity in lenti--synuclein-treated tg mice (lanes 5–8) compared to lenti-GFP-treated tg mice (lanes 1–4). (b) Compared to tg mice treated with the control lenti-GFP, tg mice treated with lenti--synuclein displayed a two-fold increase in pAkt (Ser473) immunoreactivity, whereas there is no significant difference in tAkt immunoreactivity. ^*Significant difference compared to h-synuclein tg mice treated with lenti-GFP (P<0.05, one-way ANOVA post hoc Dunnett's).

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Discussion

The present study showed that -synuclein gene transfer into a tg mouse model of PD and DLB resulted in decreased accumulation of h-synuclein in synapses and neuronal cell bodies and ameliorated the neurodegenerative alterations associated with this process. To the best of our knowledge, this is the first study to use an antiaggregation gene therapy approach for the experimental treatment of these synucleopathies. These results are consistent with in vivo studies in tg mice showing that overexpression of -synuclein (under the control of the thy-1 promoter) reduced the neurotoxic effects of -synuclein.²¹ These findings also support in vitro studies showing that -synuclein inhibited the conversion of A53T -synuclein protofibrils to fibrils³¹ and reduced the formation of -synuclein aggregates.^{21, 32} Several mechanisms might be at play, including the possibility that -synuclein might block further aggregation by preventing interactions of -synuclein with fatty acids in the membrane, favoring the -helix rather than the -pleated conformation of -synuclein.³³ In support of this, previous studies have demonstrated that - and -synuclein coimmunoprecipitate in double-transfected model systems^{21, 34} and the present study showed that -synuclein gene transfer reduced h-synuclein oligomerization and abnormal accumulation of insoluble aggregates in the plasma membrane. Furthermore, both in the brains of tg mice as well as in the in vitro model system, -synuclein coimmunoprecipitates with h-synuclein, suggesting that interactions between these two molecules might interfere with the accumulation of h-synuclein oligomers in the membrane.

Thus, -synuclein may exist in at least two different structural forms: a cytosolic unfolded form and a membrane-bound form with a stable -helical structure.^{35, 36} It has been proposed that the membrane-bound form might have a higher tendency to form aggregates than the cytosolic form and might seed the aggregation of the cytosolic -synuclein.^{35, 37} The precise mechanisms involved in this process are not completely clear; however, some studies have proposed that this might be related to the high content of polyunsaturated fatty acids in the neuronal plasma membrane.^{38, 39} -Synuclein has several fatty acid binding domains and the association of these with other molecules of -synuclein promotes aggregation.³⁸ Thus, it is possible that the binding of -synuclein to -synuclein in the cytoplasm might block aggregation by inhibiting translocation of -synuclein into the plasma membrane and interactions with fatty acids.

Alternatively, -synuclein might decrease -synuclein aggregation and neurotoxicity through indirect mechanisms, including regulation of signaling pathways that promote neuronal survival and synaptic plasticity. Among them, the present study showed in tg mice lenti--synuclein treatment increased the activity of Akt, a signaling molecule that promotes neuroprotection.⁴⁰ Furthermore, recent studies in transfected neurons have shown that lenti--synuclein protects from the combined neurotoxic effects of h-synuclein and rotenone by increasing the activation state of Akt.³⁰ The mechanisms of neuroprotection appear to be independent of upstream effects such as phosphatidylinositol 3-kinase (PI3-K) and phosphoinositide-dependent kinase (PDK)-1 and involve direct binding of -synuclein to Akt, resulting in increased stability of the kinase and phosphorylation of downstream signaling molecules such as glycogen synthase kinase 3 (GSK3)³⁰ and p53.⁴¹ In regard to the latter signaling pathway, a recent study showed that -synuclein protects from the toxic effects of 6-hydroxydopamine by decreasing the expression of the proapoptotic gene p53.⁴¹ The mechanisms linking the effects of -synuclein to the Akt and p53 signaling pathways are not completely clear. However, recent studies have shown that p53 expression and activity are regulated by Mdm2,⁴² an oncoprotein that under normal conditions is restricted to the cytoplasm but when phosphorylated by activated Akt is translocated to the nucleus. In the nucleus, phosphorylated Mdm2 binds p53, inhibiting its apoptotic activity and promoting cell survival.⁴² Taken together, these studies suggest that -synuclein-mediated activation of the Akt signaling pathway might induce phosphorylation of, among other targets, Mdm2, promoting neuroprotection against toxins by inactivating p53.

Consequently, the development of new treatments for PD and DLB might require not only promoting regeneration of the dopaminergic system but also preventing further damage mediated by the accumulation of the -synuclein neurotoxic species. Transfer of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) into the CNS of acute models of parkinsonism (ie 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine [MPTP], 6-hydroxydopamine) utilizing lentiviral vectors have recently been proposed as an alternative therapeutic approach for rescuing degenerating neurons and promoting regeneration of the NS system.^{2, 43} The present study further supports the usefulness of this strategy and extends the validity of this approach in a tg mouse model of PD and DLB by showing that gene transfer therapy might also be utilized to reduce accumulation of neurotoxic -synuclein in the brain.

In summary, these data further support a role for -synuclein in regulating the conformational state of -synuclein and suggest that this antiaggregation gene transfer approach might have potential for the development of alternative therapies for PD and DLB.

Materials and methods

Cell culture and medium

Rat neuroblastoma B103 cells were maintained in DMEM with 10% fetal calf serum (FCS) with sodium pyruvate and gentamicin. They were stably transfected with the mammalian expression vector pCEP4 (Invitrogen, Carlsbad, CA, USA) containing the h-synuclein cDNA using Lipofectamine according to the manufacturer's protocol (Gibco/BRL, Grand Island, NY, USA) as previously described.⁴⁴ This line was selected because overexpression of h-synuclein in these cells results in abnormal accumulation of h-synuclein oligomers in the neuronal cell bodies, reduced neurite plasticity and neurodegeneration.⁴⁴

Lentiviral vector production

Vector plasmids were constructed for the production of third-generation lentiviral vectors that expressed the -synuclein gene. The human cytomegalovirus (CMV) promoter was used to drive expression of the transgenes (Figure 1a). Vector plasmids were also constructed expressing the enhanced GFP. Additional control experiments were performed with an empty lenti vector control. All vectors used the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) 3' to the transgene. We produced lentiviral vectors using a four-plasmid transfection system, as previously described.⁴⁵ Briefly, 293T cells were transfected with vector and packaging plasmids, the supernatants were collected and vectors were concentrated by centrifugation. The lentiviral vector titers were estimated by measuring the amount of HIV p24 gag antigen with an ELISA kit (Perkin-Elmer, Wellesley, MA, USA) (100 000 transducing units (TU) per nanogram of p24).

Generation and transduction of stably transfected cell lines

The B103 neuroblastoma cells, vector transfected and h-synuclein transfected as previously described,⁴⁴ were grown in six-well plates at 50% confluence and were incubated with either lenti--synuclein or lenti-GFP (each at 1.0 10⁷ TU) in 10% FCS for 24 h at 37°C, 5% CO₂. The cells were then washed with phosphate-buffered saline (PBS) and incubated in DMEM with 10% FCS for an additional 4 days. The efficiency of transduction of lenti-GFP was more than 90%. Finally, cells were harvested in lysis buffer and used for immunoblot analysis. For immunocytochemistry, cells were cultured on coverslips until 50% confluence and treated as described above, fixed in 4% paraformaldehyde (PFA) for 20 min, and blocked overnight at 4°C in 10% FCS and 5% bovine serum albumin (BSA).

In vitro immunoblot (Western blot) analysis

For the detection of h- and -synuclein in vitro, transduced cells (1.0 ng p24/cell) were lysed in cell lysis buffer plus protease inhibitors (Complete™ mini, Roche Biochemicals, Indianapolis, IN, USA). The lysates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% Tris-acetate polyacrylamide gel (NuPAGE™, Invitrogen). Immunoblots were performed with primary antibodies against -synuclein (Syn-1, 1:1000, Transduction Laboratories, San Diego, CA, USA) or -synuclein (1:1000, Chemicon, Temecula, CA, USA) and a secondary goat anti-mouse or rabbit IgG tagged with horseradish peroxidase (HRP, 1:5000, 2 h, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), visualized by enhanced chemiluminescence (ECL, Perkin-Elmer) and analyzed with the VersaDoc gel imaging system (BioRad, Hercules, CA, USA).

Transgenic mouse lines and intracerebral injections of lentiviral vectors

For this study, mice overexpressing h-synuclein from the platelet-derived growth factor (PDGF-) promoter (Line D) were utilized. This model was selected because mice from this line develop h-synuclein immunoreactive inclusions distributed through the neocortex and hippocampus similar to what has been described in DLB.^{46, 47, 48} A total of 48 h-synuclein tg mice from line D (12 months old) were injected with 3 l of the lentiviral preparations (1.5 10⁷ TU) into the temporal cortex and hippocampus. Briefly, as previously described,⁴⁵ mice were placed under anesthesia on a Koft stereotaxic apparatus and coordinates (AP –1.5 mm, lateral 1 mm, depth 2 mm) were determined as per the Franklin and Paxinos Atlas. The lentiviral vectors were delivered using a 5 l Hamilton syringe connected to a hydraulic system to inject the solution at a rate of 0.5 l every 2 min. To allow diffusion of the solution into the brain tissue, the needle was left for an additional 5 min after the completion of the injection. Mice received unilateral injections (right side) to allow comparisons against the contralateral side, with either lenti--synuclein (n=24), heat-inactivated lenti--synuclein (n=6), lenti-GFP (n=10) or vehicle alone (n=10). Mice survived for 4 weeks after the lentiviral injection. Following NIH guidelines for the humane treatment of animals, mice were anesthetized with chloral hydrate and flush-perfused transcardially with 0.9% saline. Brains and peripheral tissues were removed and divided sagitally. The right hemibrain was postfixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 h for neuropathological analysis, while the left hemibrain was snap-frozen and stored at -70°C for subsequent RNA and protein analysis.

RNA analysis

Total RNA was isolated from snap-frozen tissues using the TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). The following ³²P-labeled antisense riboprobes were used to identify specific mRNAs (protected nucleotides (GenBank accession number)): -synuclein (nt 210–475 (No. L08850)); -synuclein (nt 235–459 (No. S69965)); and murine actin (nt 480–559 (No. M18194) of mouse actin mRNA). Levels of specific RNAs were determined by RPA, as previously described.⁴⁹ Dried gels were exposed to Biomax^® film (Kodak, Rochester, NY, USA) and signals were quantitated with a PhosphorImager SF (Molecular Dynamics, Sunnyvale, CA, USA) using the ImageQuant software and expressed as integrated pixel intensities over defined volumes. Final values were expressed as ratios of (specific signal-background)/(actin signal-background) to correct for differences in RNA content and loading across samples.

In vivo immunoblot (Western blot) analysis

For detection of h-synuclein in the brain, homogenates were separated into cytosolic and particulate fractions.²⁷ A measure of 12 g of each fraction per mouse was loaded onto 10% SDS-PAGE gels, followed by transfer onto Immobilon membranes. These were then incubated with the anti--synuclein antibody (Syn-1, 1:1000, Transduction Laboratories)²¹ and a secondary goat anti-mouse tagged with HRP (1:5000, 2 h, Santa Cruz Biotechnology, Inc.), visualized by ECL (Perkin-Elmer) and analyzed with the VersaDoc system (BioRad).

Coimmunoprecipitation of h-synuclein with -synuclein in transduced cells and tg mice

Coimmunoprecipitation assays were performed as previously described with minor modifications.²¹ Briefly, cell extracts (200 g) of either cytosolic or particulate fractions were diluted with 2 lysis buffer (1 lysis buffer: 1% Triton-X, 10% glycerol, 50 mM HEPES (pH 7.4), 140 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM phenyl-methyl-sulfonylfluoride, 5 g/ml aprotinin, 5 g/ml leupeptin, 1 mM dithiothreitol) and precleared with protein G-sepharose for 30 min on ice. The samples were then incubated with either pre-immune serum or anti--synuclein serum overnight at 4°C, followed by incubation with protein G-sepharose for 2 h. The immune complexes were then washed three times with the lysis buffer. The samples were then heated in the SDS sample buffer for 5 min and subjected to immunoblot analysis and visualization with ECL (Perkin-Elmer) on the VersaDoc system (BioRad).

Immunoblot analysis of Akt expression in mice treated with lenti--synuclein

Immunoblot analysis was performed as previously described.³⁰ Briefly, tg mouse brain homogenates were prepared, solubilized in lysis buffer (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 20 mM b-glycerophosphate, and proteinase inhibitor cocktails), and centrifuged for 10 min at 14 000 rpm. The supernatant (20 g) was then resolved by SDS-PAGE and electroblotted onto nitrocellulose membrane (Schleicher & Schunell, Keene, NH, USA). The membranes were blocked with Tris-buffered saline (TBS; 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 0.2% Tween-20) containing 3% skim milk or BSA, followed by incubation with primary antibodies against pAkt (Ser473, 1:1000, Cell Signaling Technology, Beverley, MA, USA) or total (t) Akt (88-10, 1:1000, Calbiochem, San Diego, CA, USA) in TBS. After washing with TBS, the membranes were analyzed with ECL (Perkin-Elmer) on the VersaDoc gel imaging system (BioRad).

Immunocytochemical and neuropathological analyses

Immunocytochemical analysis for h-synuclein was performed in serially sectioned, free-floating, blind-coded vibratome sections from tg and nontg mice treated with lenti--synuclein and control vectors.²⁷ Sections were incubated overnight at 4°C with an anti-h-synuclein antibody (72-10, 1:500, affinity purified rabbit polyclonal),²⁷ followed by biotinylated goat anti-rabbit IgG (1:100, Vector Laboratories, Inc., Burlingame, CA, USA), Avidin D-HRP (1:200, ABC Elite, Vector), and reacted with diaminobenzidine tetrahydrochloride (DAB) containing 0.001% H₂O₂. Sections were analyzed with the Quantimet 570C (Leica) in order to determine the number of h-synuclein immunoreactive inclusions in the neocortex. For each case, three sections were analyzed and the results were averaged and expressed as numbers per sq mm.

To determine efficiency of transduction, double-labeling experiments with -synuclein and the neuronal marker NeuN (1:5000, Chemicon) were performed, as previously described.²¹ To determine the relationship between h-synuclein immunolabeled-inclusions and -synuclein immunoreactive neurons, 40 m-thick vibratome sections were immunolabeled with the rabbit polyclonal antibodies against h-synuclein (72-10, 1:5000, affinity purified polyclonal)²⁷ and -synuclein (1:100, Chemicon). The h-synuclein immunoreactive structures were detected with the Tyramide Signal Amplification™-Direct (Red) system (1:100, NEN Life Sciences, Boston, MA, USA) while -synuclein was detected with the goat anti-rabbit fluorescein isothiocyanate (FITC) antibody (1:75, Vector). This system allows the simultaneous detection of signals from antibodies from the same species. To determine if -synuclein gene transfer ameliorated the neurodegenerative alterations associated with the expression of h-synuclein, sections were double-immunostained with a polyclonal antibody against h-synuclein (72-10, 1:100, affinity purified rabbit) and a monoclonal antibody against synaptophysin (SY38, 1:25, Chemicon), followed by a mixture of horse anti-mouse FITC tagged secondary antibody (1:75, Vector) to detect synaptophysin and a goat anti-rabbit Texas Red tagged secondary antibody (1:75, Vector) to detect h-synuclein. For each case, sections were immunolabeled in duplicate and analyzed with the laser confocal scanning microscope (LSCM) and NIH Image 1.43 software to calculate the percent area of the neuropil covered by synaptophysin-immunoreactive terminals in the neocortex.⁵⁰ For each case, three sections were analyzed, and for each section four serial optical sections (2 m thick) were obtained. In order to confirm the specificity of the primary antibodies, control experiments were performed where sections were incubated overnight in the absence of primary antibody (deleted), with the primary antibody preadsorbed for 48 h with 20-fold excess of the corresponding peptide or with pre-immune serum.

All sections were processed simultaneously under the same conditions and experiments were performed twice in order to assess the reproducibility of results. Sections were imaged with a Zeiss 63X (N.A. 1.4) objective on an Axiovert 35 microscope (Zeiss, Germany) with an attached MRC1024 LSCM system (BioRad, Wattford, UK).²⁷

Statistical analysis

All in vivo and in vitro experiments were conducted in triplicate on blind-coded samples. After the results were obtained, the code was broken and data were analyzed with the StatView 5.0 program (SAS Institute, Inc., NC, USA). Differences among means were assessed by one-way ANOVA followed by post hoc Dunnett's or Tukey–Kramer as indicated. Correlation studies were carried out by simple regression analysis. The null hypothesis was rejected at the 0.05 level.

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Acknowledgements

This work was supported by National Institutes of Health Grants AG5131, and AG18440 and by a grant from the MJ Fox Foundation for Parkinson's Research to EM and by AG08514 to FHG. RAM was supported in part by funds from the Canadian Institutes of Health Research.