AAV Transduction of Dopamine Neurons With Constitutively Active Rheb Protects From Neurodegeneration and Mediates Axon Regrowth

© The American Society of Gene & Cell Therapy

original article

AAV Transduction of Dopamine Neurons With Constitutively Active Rheb Protects From Neurodegeneration and Mediates Axon Regrowth Sang Ryong Kim1, Tatyana Kareva1, Olga Yarygina1, Nikolai Kholodilov1 and Robert E Burke1,2 1 Department of Neurology, Columbia University, New York, New York, USA; 2Department of Pathology and Cell Biology, Columbia University, New York, New York, USA

There are currently no therapies that provide either protection or restoration of neuronal function for adultonset neurodegenerative diseases such as Parkinson’s disease (PD). Many clinical efforts to provide such benefits by infusion of neurotrophic factors have failed, in spite of robust effects in preclinical assessments. One important reason for these failures is the difficulty, due to diffusion limits, of providing these protein molecules in sufficient amounts to the intended cellular targets in the central nervous system. This challenge suggests an alternative approach, that of viral vector transduction to directly activate the intracellular signaling pathways that mediate neurotrophic effects. To this end we have investigated the ability of a constitutively active form of the GTPase Rheb, an important activator of mammalian target of rapamycin (mTor) signaling, to mediate neurotrophic effects in dopamine neurons of the substantia nigra (SN), a population of neurons affected in PD. We find that constitutively active hRheb(S16H) induces many neurotrophic effects in mice, including abilities to both preserve and restore the nigrostriatal dopaminergic axonal projections in a highly destructive neurotoxin model. We conclude that direct viral vector transduction of vulnerable neuronal populations to activate intracellular neurotrophic signaling pathways offers promise for the treatment of neurodegenerative disease. Received 15 February 2011; accepted 12 September 2011; published online 18 October 2011. doi:10.1038/mt.2011.213

Introduction The discovery of neurotrophic factors offered the promise that these molecules, with their diverse prosurvival and growth effects, may provide effective neuroprotective or restorative therapies for neurodegenerative diseases.1,2 The pursuit of clinical benefit by use of neurotrophic factor therapy has been especially vigorous in the assessment of glial cell line-derived neurotrophic factor in the treatment of Parkinson’s disease (PD).3 Originally discovered on the basis of its ability to support developing dopamine neurons,4 a neuronal population predominantly affected in PD, glial cell line-derived neurotrophic factor was demonstrated in numerous

preclinical assessments to provide both neuroprotection and restoration in models of the disease5–7 including primate models.8 While an initial small, open trial of direct brain infusion of glial cell line-derived neurotrophic factor appeared to offer promise,9 a subsequent larger, blinded trial detected no benefit and raised concerns about off-target adverse effects.10,11 Discouraging results have also occurred in clinical trials of other neurotrophic factors in the treatment of other neurologic disorders.2 While there are many possible reasons for these failures, there is a consensus that the delivery of neurotrophic proteins to their desired neuronal targets within the central nervous system is a challenge, and efforts to overcome diffusion limits have met with off-target adverse effects.2,11 This challenge of providing protein molecules by diffusion to their appropriate cell surface receptor targets suggests on alternative approach, that of direct intracellular activation of neurotrophic cell signaling by use of viral vector-mediated transduction. In the case of glial cell line-derived neurotrophic factor, diverse cellular signaling pathways play a role,12 and the most compelling evidence has identified a major role for Ret tyrosine kinase activation of PI3K/Akt signaling.13–16 We have demonstrated that transduction of dopamine neurons of the substantia nigra (SN) with a constitutively active form of Akt, myristoylated-Akt (Myr-Akt), induces an array of neurotrophic effects in these neurons, including hypertrophy, increased expression of neurotransmitter synthetic enzymes, axon sprouting, and resistance to neurotoxin-induced cell death17 and axon degeneration.18 While these observations provide a compelling proof-of-concept, that an adeno-associated virus (AAV) vector in current clinical use19 can be used to mediate neurotrophic effects by cellular transduction, further investigation with a specific focus on the pathways that mediate clinically important phenotypic effects is needed. In PD, for example, there is a growing consensus that it is the axons of dopamine neurons, not their cell bodies, that are predominantly involved at disease onset, and, most importantly, it is progressive axon loss, not cell body loss, that determines the course of clinical progression (reviewed in ref. 20). We therefore sought to further investigate downstream mediators of PI3K/Akt signaling that have been identified as especially important to the neurobiology of axon growth and maintenance. One such mediator, the mTor kinase, has been shown to participate in many such aspects,

Correspondence: Robert E Burke, Department of Neurology, Room 306, Black Building, Columbia University, 650 West 168th Street, New York, New York 10032, USA. E-mail: [email protected] Molecular Therapy vol. 20 no. 2, 275–286 feb. 2012

275

© The American Society of Gene & Cell Therapy

Rheb Mediates Neuroprotection and Restoration

including axon growth, axon number per neuron, branching, caliber and growth cone dynamics.21–24 We have therefore investigated the effects of activation of the mTor complex 1 (mTORC1) by a principal upstream regulator, the GTPase ras homolog enriched in brain (Rheb). We have selected Rheb for this purpose because mTor is its principal downstream effector25 and well-characterized constitutively active forms exist.26–28 For these investigations, we have evaluated two mutants. Rheb(N153T) was identified following a screen in yeast that demonstrated a hyperactivating mutation in the homologue Rhb1.26,29 This mutation diminishes binding of GDP, thereby increasing the ratio of active GTP-bound to inactive GDP-bound Rheb. hRheb(S16H) was identified by a mutation

analysis that predicted and confirmed the critical nature of the serine at the 16 position for sensitivity to tuberous sclerosis complex (TSC) GTPase activation.27 Mutation of the serine to histidine results in resistance of hRheb to this activation and consequently a persistence of the GTP-bound, activated state. We have assessed the ability of wild-type hRheb and these constitutively active mutants to mediate trophic effects on SN dopamine neurons with particular attention to effects on axons. To evaluate neuroprotective and restorative effects, we have used a neurotoxin model in which 6-hydroxydopamine (6-OHDA) is injected into the striatal target of the nigrostriatal dopaminergic projection.30 The advantage of this model is that it induces a retrograde degeneration of

WT

SNpc

250 µm

N153T

SNpc

S16H

SNpc

50 µm TH

FLAG

Merge

20 µm WT

N153T

S16H

Figure 1 Transduction of SNpc neurons by adeno-associated virus (AAV)-Rheb vectors in normal adult male C57Bl/6 mice. The upper panels show immunoperoxidase staining for FLAG performed at 4 weeks after intranigral injection of each vector. For each vector, FLAG expression is observed as brown reaction product in the SNpc (arrows). No staining above background is observed in the SNpc on the control, noninjected side. FLAG immunostaining within the regions outlined by the squares is shown at higher magnification in the adjacent panels to the right. In the lower panels, immunofluorescence double-labeling for tyrosine hydroxylase (TH) (red) and FLAG (green) demonstrates that transgene expression is identifiable within dopamine neurons of the SNpc for each vector. Each vector was estimated to achieve efficiencies of transduction of dopamine neurons ranging from 80% in the caudal planes adjacent to the vector injection site to 60% in the rostral planes.

276

www.moleculartherapy.org vol. 20 no. 2 feb. 2012

© The American Society of Gene & Cell Therapy

Molecular Therapy vol. 20 no. 2 feb. 2012

a GFP

P4 (T3 E-BP 7/4 1 6) β− ac tin

C

E

Rheb (WT) C

E

Rheb (N153T)

Rheb (S16H)

C

C

E

E

FL

AG

β−

ac

tin

1.4 1.2 P-4E-BP1/β-actin

Transduction of SNpc dopamine neurons with AAV hRheb(WT), AAV hRheb(N153T), and AAV hRheb(S16H) was demonstrated by immunoperoxidase staining for the FLAG epitope, and by double immunofluorescence labeling for FLAG and tyrosine hydroxylase (TH) (Figure 1). Although FLAG expression in the SN was detected for each transgene by both immunoperoxidase staining and western analysis (Figure  2a), only hRheb(S16H) induced a detectable increase in phosphorylation of the mTor substrate 4E-BP1 (Figure 2a). The induced phosphorylation of 4E-BP1 in SN was demonstrated by immunoperoxidase staining to occur exclusively within neurons of the SNpc (Figure 2b). mTor signaling has been demonstrated to regulate cell size, including that of neurons, in a variety of contexts. We therefore sought to determine whether the hRheb transgenes had such effects. Among the three vectors, hRheb(S16H) had the most pronounced effect on the size of dopamine neurons of the SN, inducing a 35% increase in area (Figure 3a). Although we were unable to detect a significant effect of hRheb(N153T) on phosphorylation of 4E-BP1 in the SN, it nevertheless had a small effect on the size of SN dopamine neurons (Figure  3a). We have previously shown that a constitutively active form of Akt/PKB, an upstream activator of both Rheb and mTor signaling,17 not only increases dopamine neuron size, but also the expression of TH in the SN. Therefore, to exclude the possibility that immunodetection of TH by peroxidase staining may produce an artifactual overestimate of neuron size, we also examined effects in tissue sections stained for the general neuronal marker NeuN. Among NeuNstained neurons, hRheb(S16H) exclusively produced an increase in neuron size in the SNpc (Figure 3b). To determine whether this effect occurred in other neuronal phenotypes as well, we examined SNpr neurons, most of which are nondopaminergic. In this population, hRheb(S16H), but not hRheb(N153T), produced a significant increase in neuron size, determined by NeuN staining (Figure 3b). hRheb(S16H) increased the size not only of the cell soma, but also the nucleus. In tissue sections stained for NeuN, in which the nuclei of SNpc neurons were clearly delineated (Figure  3b), hRheb(S16H) induced a 22% increase in nuclear size in comparison to the contralateral noninjected control side (control: 48.7 ± 0.9 µm2; hRheb(S16H): 59.5 + 1.0 µm2; P < 0.001 ANOVA). Although neuronal hypertrophy is a well-recognized neurotrophic effect, its functional consequences are not well known. Of greater functional relevance are effects on axon growth and connectivity, and neurotransmitter synthesis and release. We therefore examined the effects of the hRheb transgenes on these aspects of SN dopamine neurons. Among the viral vectors, only AAV hRheb(S16H) had a significant effect on dopaminergic innervation of the striatum, assessed by immunoperoxidase staining of TH in

CON EXP

P = 0.004

1.0 0.8 0.6 0.4 0.2 0.0

GFP

WT

N153T

S16H

b CON

EXP

GFP

Results Neurotrophic effects of hRheb(S16H) on dopamine neurons of the SN in normal adult mice

dopaminergic axons and terminals (Figure 4a,b). An increase in optical density of striatal TH immunostaining at the regional level could be attributable to an increase in the number of dopaminergic axons or terminals in the striatum, but it may also be due to an increase in the amount of TH protein per axon or terminal structure. However, medially, adjacent to the nucleus accumbens, individual TH-positive axons can be discerned, because they are

Rheb (S16H)

the dopaminergic axons that is complete by 1 week,31,32 associated with a more slowly progressive loss of neurons that is complete by 8 weeks. At this late postlesion time, neuron loss is estimated to be 60–80%, comparable to the extent of loss after a 10–15 year course of PD.33

Rheb Mediates Neuroprotection and Restoration

Figure 2  Phosphorylation of the mTor substrate 4E-BP1 by hRheb(S16H). (a) Western analysis of p-4E-BP1 expression in the ventral mesencephalon at 4 weeks after intranigral injection of adeno-associated virus (AAV) vectors. The upper panel shows a representative blot, and the lower panel shows a quantitative analysis based on the density of the p-4E-BP1 bands normalized for the β-actin band for each sample. hRheb(S16H) induced a 2.6-fold increase in the p-4E-BP1/β-actin ratio in comparison to the mean ratio for the four contralateral controls (P < 0.001, oneway ANOVA; P = 0.004 Tukey post-hoc analysis; n = 4 animals, each group). Neither hRheb(WT) nor hRheb(N153T) had significant effects. Successful transduction of the substantia nigra (SN) was confirmed in each case by western analysis of FLAG expression as shown (C, control side; E, experimental side). (b) Immunoperoxidase staining for p-4E-BP1 (with thionin counterstain) in the SN at 12 weeks following transduction with AAV hRheb(S16H). Brown reaction product is observed in neurons (arrows) on the injected side, but not on the contralateral control side or on either side following AAV green fluorescent protein (GFP) injection (Bar = 100 μm). An example of neuronal staining is shown in the right panel.

277

© The American Society of Gene & Cell Therapy

Rheb Mediates Neuroprotection and Restoration

b

EXP

AAV hRheb (S16H)

CON

250 µm

CON

EXP

AAV hRheb (S16H)

a

250 µm

20 µm

20 µm

300

P < 0.05

P < 0.001

200 100 0

WT

400

TH − SNpc

CON EXP

N153T

S16H

Cell area (µm2)

Cell area (µm2)

400

300

CON EXP

NeuN − SNpr P < 0.001

200 100 0

WT

N153T

S16H

Figure 3 Effects of wild-type (WT) and constitutively active forms of hRheb on substantia nigra (SN) neurons in normal adult male C57Bl/6 mice. (a) Morphologic analysis of SN dopamine neurons at 4 weeks after intranigral injection of adeno-associated virus (AAV) hRheb(S16H). The upper panel shows a representative coronal section of the SN following tyrosine hydroxylase (TH) immunoperoxidase stain and thionin counterstain. The experimental (EXP) side injected with AAV hRheb(S16H) shows an increased density of staining. This increase is associated with an increase in the mean area of the TH-positive neurons, as shown in representative micrographs at higher power in the lower panels. This effect is shown quantitatively for mice receiving AAV Rheb(S16H) as a 35% increase in mean neuron size on the injected side in comparison to mean size on the contralateral control noninjected side (P < 0.001, one-way ANOVA and Tukey post-hoc analysis as shown; n = 80 neurons from four mice, each experimental group). AAV Rheb(N153T) also increased neuron size, by 19% (Tukey post-hoc analysis as shown; n = 80 neurons from four mice, each experimental group). (b) Morphologic analysis of SN neurons demonstrated by immunoperoxidase staining for the neuron marker NeuN. The EXP side shows an increased density of staining in the SNpc. As for the TH staining, this increase is associated with an increase in the mean cross-sectional area of the NeuN-positive neurons, as shown in representative micrographs at higher power in the lower panels. This effect on neuron size is general to other neuron phenotypes as shown quantitatively in the graph, which reveals a 42% increase in the size of NeuN-positive neurons in the SNpr (P < 0.001, one-way ANOVA and Tukey’s post-hoc analysis as shown).

not obscured by the dense plexus of TH-positive fibers present in the striatum (Figure 4a). In this location, hRheb(S16H) induced an increased number of TH-positive axons. hRheb(S16H) also induced an increase in the density of immunostaining for the dopamine transporter (DAT), a membrane-bound protein localized to nerve terminals (Figure 4b). These morphologic changes induced by hRheb(S16H) were associated with correlated neurochemical changes. Both dopamine and homovanillic acid (HVA), a major metabolite, were increased in the striatum following transduction of the SN with AAV hRheb(S16H) (Figure 4c). Most importantly, and in keeping with the possibility that hRheb(S16H) had induced functional axonal sprouting in the striatum, transduction with AAV hRheb(S16H) induced an altered behavioral response to treatment with amphetamine. Following intranigral injection of AAV hRheb(S16H) there was no apparent alteration in spontaneous motor activity at 4 weeks. However, the ability of amphetamine to induce dopamine release can be utilized to disclose an underlying imbalance between the left and right nigrostriatal projections that may not be manifested in spontaneous activity.34 In the presence of a unilateral increase in the capacity to release dopamine in the striatum, amphetamine can induce contraversive rotational behavior34 (Figure  4d). Such behavior was observed in mice injected with AAV hRheb(S16H), but not AAV green fluorescent 278

protein (GFP) controls (Figure 4d). We therefore conclude that in normal adult mice hRheb(S16H) has neurotrophic effects on the nigrostriatal dopaminergic projection that result in morphologic, neurochemical and functional alterations.

Neuroprotective effects of hRheb(S16H) on SN dopamine neurons in the unilateral 6-OHDA neurotoxin model A cardinal effect of neurotrophic factors, and one that has created the greatest interest in relation to potential for the treatment of neurodegenerative disease, is the ability to forestall neuron degeneration. Given the more pronounced effects of hRheb(S16H) in normal mice as compared to the other forms of hRheb, we evaluated its ability to protect SN dopamine neurons from neurotoxininduced death. For this evaluation we utilized a model based on intrastriatal injection of 6-OHDA,30 adapted for mice.35 This model induces retrograde degeneration of dopaminergic axons during the first postlesion week,31 with the subsequent occurrence of apoptosis in dopamine neurons,31,36 and the progressive loss of neurons over weeks.30 Transduction of SN neurons with AAV hRheb(S16H) 3 weeks before intrastriatal injection of 6-OHDA provided striking protection of dopamine neurons. Whereas only 43% survived in AAV GFP-treated mice, 90% survived in mice treated with AAV hRheb(S16H) (Figure 5b). This neuroprotection www.moleculartherapy.org vol. 20 no. 2 feb. 2012

© The American Society of Gene & Cell Therapy

Rheb Mediates Neuroprotection and Restoration

a

c CON

EXP

200

200

GFP hRheb (S16H

GFP hRheb (S16H

100

0 SN

STR

P = 0.001

TH 140 120

STR SNpc

100

R

80

L

STR

0

AMPHETAMINE

(-) CONTRAVERSIVE 160

SN

−20 NET TURNS/60 minutes

20 µm

Striatal od % CON

100

50 0

d

b

P = 0.001

150 HVA % CON

1 mm

Dopamine % CON

AAV hRheb (S16H)

P = 0.006 150

−40 −60 −80 −100 −120

P = 0.009 GFP hRheb (S16H

−140 Rotational behavior

60 20 0 GFP

WT

N153T

S16H

160

Striatal od % CON

140

DAT P < 0.001

120 100 80 60 40 20 0 GFP

WT

N153T

S16H

Figure 4 Relative effect of wild-type (WT) and constitutively active forms of hRheb on measures of striatal dopaminergic innervation in normal adult male C57Bl/6 mice. (a) Morphologic analysis of striatum at 4 weeks after intranigral injection of adeno-associated virus (AAV) hRheb(S16H). In the upper panel a representative coronal section of tyrosine hydroxylase (TH) immunoperoxidase staining reveals an increased density of staining on the experimental (EXP) side injected with AAV hRheb(S16H). As shown in the micrographs in lower panels, this increase is due in part to an increased number of TH-positive axons. The increase in optical density for TH peroxidase stain is shown quantitatively in panel (b). AAV hRheb(S16H) induced a 1.4-fold increase in optical density (OD) of TH peroxidase stain, expressed as percent of the contralateral, noninjected side, in comparison to AAV green fluorescent protein (GFP)-injected mice (P = 0.001, one-way ANOVA and Tukey post-hoc analysis as shown; n = 4 animals, each experimental group). AAV hRheb(S16H) also induced a 1.2-fold increase in optical density (OD) of peroxidase stain of the dopamine transporter (DAT) (P < 0.001, one-way ANOVA and Tukey post-hoc analysis as shown; n = 4 animals, each experimental group). (c) These morphologic effects of AAV hRheb(S16H) were accompanied by increases in biochemical measures of striatal dopaminergic innervation. In comparison to AAV GFP control injection, AAV hRheb(S16H) induced significant increases in striatal dopamine (DA) and its metabolite homovanillic acid (HVA), expressed as percent of the contralateral, noninjected control striatum (P = 0.003 and P < 0.001, respectively; t-test; n = 7 and 8, respectively). (d) To determine whether these morphologic and biochemical measures of increased striatal dopaminergic innervation were functionally significant, we administered amphetamine [2.5 mg/kg intraperitoneal (i.p.)] to mice at 4 weeks following intranigral injection of either AAV GFP or AAV hRheb(S16H). Amphetamine induces dopamine release from striatal terminals, and in the presence of a unilateral increase in functional dopaminergic innervation, it will induce a rotational behavior contralateral to the side with the increase (upper panel).34 Following AAV GFP injection, no sustained rotational behavior was observed (lower panel). However, after injection of AAV hRheb(S16H), a highly significant contralateral rotational behavior (depicted as negative on the ordinate) was observed following amphetamine injection (P = 0.009, t-test, n = 8 animals, each experimental group).

was accompanied by a modest, but significant, preservation of dopamine and its metabolite, HVA, in ventral mesencephalon (Figure 5c). Molecular Therapy vol. 20 no. 2 feb. 2012

Many molecular approaches to neuroprotection of SN dopamine neurons have demonstrated an ability to protect neuron numbers, but have been ineffective in the preservation of their 279

© The American Society of Gene & Cell Therapy

Rheb Mediates Neuroprotection and Restoration

axons.31,37 Remarkably, hRheb(S16H) demonstrated a robust preservation of the nigrostriatal dopaminergic projection at the level of the striatum. At 4 weeks postlesion mice treated with AAV GFP demonstrated only a 32% preservation of striatal TH

a

Viral injection

6−OHD A Lesion

Perfusion

3

7

Week 0

b

EXP

GFP

CON

hRheb (S16H)

250 µm

c

8000

TH positive neurons

P < 0.001

P < 0.001

CON EXP

6,000

4,000

2,000

0 GFP

S16H

5

Dopamine (ng/sample)

CON EXP

4 P = 0.08 P = 0.03

NS

3 2 1 0 GFP

S16H

5

CON EXP

HVA (ng/sample)

4 P = 0.07 P = 0.03

NS

3 2 1 0

280

GFP

immunoperoxidase staining, whereas mice treated with AAV hRheb(S16H) demonstrated 85% preservation (Figure 6a). The optical density of staining in the striatum on the lesioned side of AAV hRheb(S16H)—injected mice was not significantly different from the contralateral, nonlesioned control side. Preservation of the nigrostriatal dopaminergic projection by this morphologic assessment was accompanied by preservation of neurochemical measures. In mice treated with AAV GFP, striatal dopamine levels were reduced to just 2.5% of contralateral control values at 4 weeks postlesion (Figure 6b). This profound loss of striatal dopamine content is typical of this highly destructive model. In mice treated with AAV hRheb(S16H), striatal dopamine content was preserved to 52% of the contralateral control; a 20-fold increase. Preservation of the nigrostriatal projection by hRheb(S16H), as demonstrated by these morphologic and biochemical measures, resulted in preserved function as well, indicated by both biochemical and behavioral assessments. Following a nigrostriatal lesion, there is an increase in dopamine release by the remaining dopaminergic terminals, indicated by an increased HVA/dopamine ratio.38 This increase has been interpreted to be a compensatory response to the partial loss of dopaminergic axons. In mice treated with AAV GFP, at 4 weeks following 6-OHDA lesion, the HVA/dopamine ratio on the lesioned side was increased 7.4-fold in comparison to the contralateral control. In mice treated with AAV hRheb(S16H) there was only a threefold increase, indicating a greater degree of functional preservation of the nigrostriatal projection (Figure  6b). Following destruction of the nigrostriatal projection, administration of amphetamine, which induces dopamine release predominantly on the intact side, results in ipsiversive rotations.34 Such a behavioral response was observed in mice treated with AAV GFP (Figure 6c). However, this rotational response was not observed in mice treated with AAV hRheb(S16H), indicating that substantial nigrostriatal dopaminergic function was preserved.

S16H

Figure 5  hRheb(S16H) protects substantia nigra (SN) dopamine neurons from 6-hydroxydopamine (6-OHDA)-induced neuron death. (a) Mice received intranigral injection of adeno-associated virus (AAV) green fluorescent protein (GFP) as control or AAV hRheb(S16H) and 3 weeks later they received an intrastriatal injection of 6-OHDA. The nigrostriatal dopaminergic projection was assessed at 4 weeks following 6-OHDA. (b) Representative coronal sections of the SN following tyrosine hydroxylase (TH) immunoperoxidase stain and thionin counterstain from mice that received either AAV GFP (upper) or AAV hRheb(S16H) (lower). In the mice that received AAV hRheb(S16H), the population of TH-positive dopamine neurons in the SNpc is relatively preserved. The neuroprotective effect of hRheb(S16H) is shown quantitatively; while only 43% of dopamine neurons survived following AAV GFP, 90% survived following AAV hRheb(S16H), due to a 2.2-fold increase in the absolute number of surviving neurons (P < 0.001, one-way ANOVA and Tukey post-hoc analysis as shown; n = 7 animals, each experimental group). The number of surviving neurons in the AAV hRheb(S16H) condition was not significantly different from the contralateral, noninjected control side (P = 0.65, NS). (c) Morphologic preservation of dopamine neurons in the SN was accompanied by relative preservation of SN dopamine and its metabolite homovanillic acid (HVA). For dopamine, one way ANOVA revealed a significant difference for the comparison between AAV GFP experimental (EXP) (6-OHDA-injected side) and the AAV hRheb(S16H) noninjected control (CON) (P = 0.03), but not for the AAV hRheb(S16H) experimental (EXP) comparison with the noninjected control (CON) (P = 0.9, NS). A similar result was obtained for HVA.

www.moleculartherapy.org vol. 20 no. 2 feb. 2012

© The American Society of Gene & Cell Therapy

CON

b

EXP

80 P < 0.001

GFP

DOPAMINE (ng/sample)

60

0.30

CON EXP

0.20 0.15

0.00

STR SNpc

20

GFP

S16H

1.6 P < 0.001 P = 0.005

1.2

0.05 GFP

S16H

AMPHETAMINE

(+) IPSIVERSIVE

0

0.10

c

CON EXP

40

P < 0.001 P = 0.002

HVA/DA

hRheb (S16H)

1 mm

Optical density

0.25

P < 0.02 P < 0.02

n.s.

0.8 0.4 0.0 GFP

S16H

60HDA R

L

Rotational behavior CON EXP

P = 0.031

200 NET TURNS/60 minutes

a

Rheb Mediates Neuroprotection and Restoration

150 100 50 0 −50

GFP S16H

Figure 6  hRheb(S16H) preserves striatal dopaminergic innervation following intrastriatal 6-hydroxydopamine (6-OHDA). (a) Representative tyrosine hydroxylase (TH) immunoperoxidase stained coronal sections of the striatum from mice that received either adeno-associated virus (AAV) green fluorescent protein (GFP) (upper) or AAV hRheb(S16H) (lower) before receiving intrastriatal 6-OHDA. In the mice that received AAV hRheb(S16H), the TH-positive dopaminergic innervation of the striatum is substantially preserved. This neuroprotective effect of hRheb(S16H) is shown quantitatively as the optical density of immunoperoxidase stain measured over the entire striatum (outlined in blue). In the mice that received AAV GFP, optical density values were reduced to 32% of the control values, whereas in the mice treated with AAV hRheb(S16H), they were reduced only to 85% (P = 0.002, one-way ANOVA and Tukey post-hoc analysis as shown; n = 7 animals, each experimental group). (b) This morphologic preservation of dopamine innervation of the striatum was accompanied by relative preservation of striatal dopamine. In AAV hRheb(S16H)-treated mice (n = 8), mean striatal dopamine content was 20-fold greater than the mean value in AAV GFP-treated mice (n = 7) (P < 0.02, one-way ANOVA and Tukey post-hoc analysis as shown). Following a partial nigrostriatal lesion, there is a compensatory increase in the turnover of dopamine, indicated by an increase in the homovanillic acid (HVA)/dopamine ratio. In the AAV hRheb(S16H)-treated mice, there was a significant decrease in this ratio, signifying a partial restoration of dopaminergic innervation (P < 0.005, one-way ANOVA and Tukey post-hoc analysis as shown). (c) Following unilateral 6-OHDA lesion, administration of amphetamine induces ipsiversive rotation, due to the predominance of dopamine release on the intact side. Mice treated with AAV hRheb(S16H), unlike mice treated with AAV GFP, did not demonstrate ipsiversive rotational behavior, indicating relative preservation of nigrostriatal dopaminergic function following 6-OHDA lesion (P = 0.03, t-test,; n = 7 animals, each experimental group).

hRheb(S16H) induces regrowth of dopaminergic axons following their destruction by 6-OHDA While suppression of neuron death and axon degeneration are very desirable phenotypes in the treatment of neurodegenerative disease, in practical clinical treatment, intervention is initiated only after the diagnosis has been made, by which time extensive damage has already been done.20 We have previously shown that AAV hRheb(S16H), when administered 3 weeks after 6-OHDA lesion, is able to induce dopaminergic axon regrowth.32 However, whether hRheb(S16H) can mediate such effects at six weeks after lesion, by which time the degenerative process has run its course, has not been assessed. Such a scenario would be more representative of mid- to late-stage human PD. We therefore examined the ability of AAV hRheb(S16H) to induce axon growth at 6 weeks following 6-OHDA and assessed striatal dopaminergic innervation at 18 weeks following lesion (Figure 7a). We found that although AAV hRheb(S16H) did not in this paradigm influence the number of surviving dopamine neurons, it did achieve a significant reinervation of the medial forebrain bundle and striatum (Figure 7). Thus hRheb(S16H) is capable of inducing a robust and lasting regrowth of dopaminergic axons long after their destruction.

Discussion The mTor kinase exists in two complexes, mTORC1 and mTORC2, which play central roles in the integration of cell growth in response to environmental conditions, including growth factors, amino Molecular Therapy vol. 20 no. 2 feb. 2012

acids, energy substrates and oxygen (reviewed in refs. 39–42). mTORC2 activates Akt activity by Ser473 phosphorylation,43 whereas mTORC1 is an important mediator of many effects of Akt on cell growth that are induced by growth factors (Figure 8). The TSC, consisting of a heterodimer of TSC1 (hamartin) and TSC2 (tuberin), is a central integrator of diverse cellular signaling pathways that impinge upon mTORC1 and ultimately regulate cell growth44 (Figure 8). TSC2 is a GTPase activating protein for Rheb, and as such it negatively regulates Rheb by mediating its conversion to an inactive, GDP-bounded state.45 In its active, GTPbound state, Rheb, by diverse mechanisms that remain to be fully elucidated, activates mTORC1.25 In order to characterize effects of Rheb-mediated activation of mTORC1 on dopamine neurons, we used two constitutively active forms of hRheb, an N153T mutant that exhibits diminished binding of GDP26 and an S16H mutant that exhibits resistance to GTPase activation by TSC2.27 These mutants have not previously been studied in living brain, so in order to monitor the activation state of mTORC1 in brain, we have assessed the phosphorylation status of 4E-BP1, a principal mTORC1 substrate,28 by both biochemical and histological methods. For the two hRheb mutants, although both proteins were detected in ventral mesencephalon by FLAG expression following transduction, only hRheb(S16H) induced an increase in the tissue levels of phosphorylated 4E-BP1 detected both by Western and immunohistochemical analysis. To confirm that enhanced 4E-BP1 phosphorylation induced by hRheb(S16H) was accompanied by a 281

© The American Society of Gene & Cell Therapy

Rheb Mediates Neuroprotection and Restoration

b

a

Control

Experimental

AAV

PERFUSION

6

18

c

FR

hRheb (S16H)

60HDA WEEK 0

6,000 4,000 2,000 0

EXP

CON

CON EXP

8,000

GFP

S16H

EXP

hRheb (S16H)

GFP

CON

TH positive neurons

GFP

FR

e

Striatal OD % CON

100

60 40

P = 0.03

P = 0.05

20 0

CON EXP

250

80 MFB axons

d

200 150

P = 0.01

P = 0.05

100 50

PLT 18W

GFP Rheb (S16H)

0

PLT 18W

GFP

S16H

Figure 7 Restoration of the dopaminergic nigrostriatal projection by AAV hRheb(S16H) at 6 weeks following 6-hydroxydopamine (6-OHDA) lesion. (a) To assess the ability of hRheb(S16H) to restore the dopaminergic projection after injury, mice were first lesioned unilaterally by intrastriatal 6-OHDA injection, and then at 6 weeks postlesion, by which time degenerative processes are complete, the substantia nigra (SN) was transduced with AAV hRheb(S16H) or green fluorescent protein (GFP) control. Twelve weeks after adeno-associated virus (AAV) (18 weeks postlesion) mice were sacrificed for morphological assessment. (b) hRheb(S16H) transduction at 6 weeks postlesion does not affect the number of surviving SN TH-positive neurons, as shown by representative coronal sections of the SN (FR: fasciculus retroflexus) or by stereology counts as shown in the graph. In mice treated with AAV GFP control injection, the number of TH-positive neurons was reduced to 2237 ± 399 by 6-OHDA, a value 31% of the nonlesioned contralateral SN. In mice treated with AAV hRheb(S16H), TH-positive neurons were reduced to 1,925 ± 451, or 28%, of the nonlesioned contralateral control. (c) In mice treated with hRheb(S16H), there was a significant reinervation of the striatum, as shown by peroxidase staining for TH. In mice treated with AAV hRheb(S16H) (N = 4), striatal peroxidase staining on the lesioned side, measured as optical density over the entire striatum (outlined in blue), was 44.6 ± 0.4% of the contralateral control. In AAV GFP-injected mice (N = 4), it was 24.6 ± 3.0%, and in non-AAV-injected mice (N = 4), it was 26.6 ± 8.2%, both significantly different from AAV hRheb(S16H) [P = 0.04, ANOVA; Student–Newman–Keuls, as shown in (d)]. In addition, mice treated with AAV hRheb(S16H) showed an increased number of TH-positive axons in the medial forebrain bundle (MFB). These mice (N = 3) had a mean of 140 ± 5.6 axons on the lesioned side (71% of the contralateral control), whereas AAV GFP (N = 3) and non-AAV-injected mice (N = 4) had only 91.5 ± 12.9 (49%) and 84.9 ± 9.6 (43%) axons, respectively, a highly significant difference [P < 0.001, ANOVA; Tukey post-hoc comparisons as shown in (e)].

282

www.moleculartherapy.org vol. 20 no. 2 feb. 2012

© The American Society of Gene & Cell Therapy

GDP

Rheb Mediates Neuroprotection and Restoration

PDK1

GTP

GDP

PDK1

GTP

TSC1

TSC1

RHEB (WT)

A K T

TSC2

mTORC1

4E-BP1

T308

RHEB (S16H)

A K T

TSC2

S473

mTORC2

S6K

mTORC1 Effects: Cell growth; Axon growth; Anti-apoptosis; Anti-macroautophagy

hRHEB (WT)

S473

mTORC2

mTORC1

4E-BP1

T308

S6K

mTORC1 Effects: Cell growth; Axon growth; Anti-apoptosis; Anti-macroautophagy

hRHEB (S16H)

Figure 8  Schematic representation of Akt/Rheb/mTor signaling pathways. Following activation at the plasma membrane by phosphorylation by PDK1 and mTORC2, Akt phosphorylates and thereby inhibits the GTPase activity of the tuberous sclerosis complex (TSC). This inhibition allows accumulation of activated GTP-bound Rheb, which is a principal activator of the mTORC1 kinase. Two principal downstream substrates of mTORC1 are 4E-BP1 and p70S6K. Their phosphorylation mediates effects of mTORC1 activation (see reviews in refs. 41,42,44). Constitutively active hRheb(S16H) is resistant to GTPase activation by TSC, and it therefore maintains enhanced activation of mTORC1.

biologic effect of mTORC1 activation, we assessed neuron size. As predicted, hRheb(S16H) expression did induce an increase in neuron size, by 35%. We therefore conclude, based on increased phosphorylation of a principal substrate, and observation of a well-characterized biologic effect, that hRheb(S16H) expression achieved activation of mTORC1. In addition to direct interaction with and activation of mTor, hRheb also interacts with FKBP38 and phospholipase D1.25 Both of these interactions are postulated to also activate mTORC1. Thus, while other effectors of hRheb signaling may remain to be identified, at present it is reasonable to postulate that the cellular phenotypes induced by hRheb(S16H) are mediated through mTORC1 signaling. In addition to an increase in the size of mesencephalic dopamine neurons of normal adult mice, hRheb(S16H) had morphologic and biochemical effects on their axons. These effects included an increase in the optical density of immunoperoxidase staining for both TH and DAT in the striatum, and increased striatal content of dopamine and one of its principal metabolites, HVA. We found that these changes were associated with an increase in amphetamine-inducible contraversive rotations, indicating a functionally significant increase in releasable dopamine. These morphologic, biochemical and behavioral changes could be attributable to new dopaminergic axon growth induced by hRheb(S16H), or, alternatively, to an augmentation of the dopaminergic phenotype within preexisting axons, resulting in increased dopamine release capability. We attempted to distinguish between these two possibilities by making observations at the fiber level medial to the nucleus accumbens, where individual Molecular Therapy vol. 20 no. 2 feb. 2012

fibers are not obscured by a dense fiber plexus, as they are in the striatum. In this location, we observed an increased number of TH-positive axons, suggesting that axon sprouting had occurred. Nevertheless, we acknowledge that detection of axon fibers by immunoperoxidase staining can be influenced by the level of expression of phenotype proteins, such as TH. Ultimately, resolution of this issue will require direct approaches to the detection of axon sprouting. In addition to these effects on normal adult dopamine neurons, hRheb(S16H) demonstrated a robust ability to protect them from 6-OHDA-induced neuron death. Its ability to reduce neuron loss was comparable to the degree of protection that we had previously observed with Myr-Akt.17 This degree of protection by hRheb was not predicted, because Akt, in addition to phosphorylation and inhibition of TSC2, also inhibits numerous other apoptotic pathways that are not known to be regulated by mTORC1 signaling.46–48 Nevertheless, mTor activation does have well-characterized antiapoptotic effects,49 and in this model, activation of mTORC1 signaling by hRheb(S16H) appears to be sufficient to replicate the acute neuron protection provided by Myr-Akt. hRheb(S16H) demonstrated an ability to preserve dopamine axons as well as cell bodies. This axonal preservation should not be simply attributed to cell body preservation, with secondary axonal preservation, because many prior investigations in this and other neurotoxin models have demonstrated that preservation of neuron cell bodies alone is not sufficient to provide axon protection.31,37 There are two fundamentally distinct mechanisms 283

Rheb Mediates Neuroprotection and Restoration

whereby hRheb(S16H) may have provided the preservation of dopaminergic axons observed at 4 weeks postlesion. First, this preservation may have been due to a neuroprotection against acute retrograde axon degeneration induced by this model. Indeed, we have reported that both Myr-Akt and hRheb(S16H) are capable of providing such protection acutely following neurotoxin injection.18 Alternatively, axon degeneration may ultimately have taken place over the course of the four postlesion weeks, but following this axon loss, new axon growth from remaining neurons may have been induced. Such a possibility is in keeping with our proposal that hRheb induces sprouting in normal adult dopamine neurons. Furthermore, in direct support of this possibility, we herein show that transduction of dopamine neurons of the SN with AAV hRheb(S16H) at 6 weeks after intrastriatal 6-OHDA lesion, a robust axon regrowth response is induced. Thus, in this model, the axon preservation could be due to a combination of axon neuroprotection and regrowth effects. Whichever of these two mechanisms plays the principal role in preserving the dopaminergic axon projection, the effect is robust and functionally significant, because it results in suppression of the ipsiversive rotational response that occurs after 6-OHDA lesion. Prior investigations of this behavioral response in rats have indicated that it will occur only after striatal dopaminergic innervation has been reduced by 30% or more.50 These observations would suggest that transduction of dopamine neurons with AAV hRheb(S16H) restores innervation to levels to 70% or more of control. This behavioral result is consistent with our quantitative morphological analyses that indicated preservation of striatal innervation at 85% of the nonlesion control values. In conclusion, we find that hRheb(S16H) has robust and functionally significant trophic effects on the dopaminergic nigrostriatal projection in normal animals and in lesioned animals by providing both neuroprotective and restorative effects. These effects are comparable to those previously observed with a constitutively active form of Akt.17 We therefore conclude that mTORC1 signaling is sufficient to recapitulate the therapeutically relevant effects of Akt on the axons of dopamine neurons. While our previous observations for neurotrophic effects of Akt17,18,32 and our present observations for hRheb establish an important proof-of-concept that these molecules mediate potentially therapeutic effects, neither can be used directly for human gene therapy because they are both potent oncogenes. However, our present observations suggest that it may be possible to circumvent this adverse effect by exploring signaling pathways downstream to mTORC1.

Materials and Methods Production of AAV viral vectors. All vectors used for these studies were AAV1 serotype. A plasmid carrying the human ras homolog enriched in brain (hRheb) was purchased from OriGene Technologies (Rockville, MD). hRheb DNA was amplified and modified to incorporate a FLAG-encoding sequence at the 3′-end by expanded long-template PCR (Roche, Indianapolis, IN). Constitutively active mutants of hRheb (N153T and S16H) were generated by use of the Phusion Site-directed Mutagenesis Kit of New England Biolabs in the pGEM-T vector (Promega, San Luis Obispo, CA). Genetically modified forms of hRheb were then cloned into an AAV packaging construct that utilizes the chicken β-actin promoter, and contains a 3′ WPRE (pBL). All nucleotide sequences in the AAV packaging constructs were confirmed

284

© The American Society of Gene & Cell Therapy

before AAV production. AAVs were produced by the University of North Carolina Vector Core. The genomic titer of AAV wild-type hRheb(WT) was 3 × 1012 viral genomes/ml, and those of hRheb(N153T) and hRheb(S16H) were 2 × 1012 viral genomes/ml. Enhanced GFP, used as a control, was subcloned into the same viral backbone, and viral stocks were produced at a titers of 2.0 × 1011 and 6 × 1012 viral genomes/ml. Intranigral AAV injection. Adult (8 week) male C57BL/6 mice were obtained from Charles River Laboratories, Wilmington, MA. Mice were anesthetized with ketamine/xylazine solution and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA) with a mouse adapter. The tip of 5.0 μl syringe needle (26S) was inserted to stereotaxic coordinates AP: −0.35 cm; ML: +0.11 cm; DV: −0.37 cm, relative to bregma. Viral vector suspension in a volume of 2.0 μl was injected at 0.1 μl/minute over 20 minutes. 6-OHDA lesion. Mice were first pretreated with desipramine to block

6-OHDA uptake by noradrenergic terminals, thereby limiting the lesion to striatal dopaminergic terminals. They were then anesthetized with ketamine/xylazine solution, and placed in a stereotaxic frame. A solution of 6-OHDA (5.0 µg/µl in 0.9% NaCl/0.02% ascorbate) was injected by microliter syringe at a rate of 0.5 µl/minute by pump for a total dose of 15.0 µg/ 3  µl. The injection was performed into the left striatum at coordinates AP: +0.09 cm; ML: +0.22 cm; DV: −0.25 cm relative to bregma. After a wait of 2 minutes, the needle was withdrawn slowly.

Institutional review of animal protocols. All injection procedures, as

described above, were approved by the Columbia University Animal Care and Use Committee.

Immunohistochemical staining procedures. For TH immunostaining, mice were perfused through a cannula placed in the left ventricle with 0.9% NaCl followed by 4.0% paraformaldehyde in 0.1 mol/l phosphate buffer, pH 7.1. The brain was carefully removed and blocked into midbrain and forebrain regions. The region containing the midbrain was postfixed for 1 week, cryoprotected in 20% sucrose overnight, and then rapidly frozen by immersion in isopentane on dry ice. A complete set of serial sections was then cut through the SN at 30 μm. Beginning with a random section between 1 and 4, every fourth section was processed, in keeping with the fractionator method of sampling (see below). Sections were processed freefloating. The primary antibody was rabbit anti-TH (Calbiochem, La Jolla, CA) at 1:750. Sections were then treated with biotinylated protein A and avidin-biotinylated horseradish peroxidase complexes (ABC; Vector Labs, Burlingame, CA). After immunoperoxidase staining, sections were thionin counterstained. The forebrain region containing the striatum was postfixed for 48 hours, frozen without cryoprotection, and processed as described previously.51 Immunostaining for DAT was performed with rat anti-DAT (Chemicon, Temecula, CA) at 1:1,000, and for neuron-specific nuclear protein (NeuN) with a mouse monoclonal antibody at 1:100 (Chemicon). Sections were incubated with biotinylated anti-rat or anti-mouse IgG (Vector Labs), respectively, followed by ABC (Vector Labs). For immunostaining of the FLAG epitope, sections were initially treated with Mouse-onMouse Blocking Reagent (Vector Labs) and processed free-floating with a mouse monoclonal anti-FLAG antibody (Sigma, St Louis, MO) at 1:1,000. Sections were incubated with biotinylated anti-mouse IgG (Vector Labs), followed by ABC (Vector Labs). For immunofluorescent staining, fluorescein conjugated avidin was used after secondary antibody. Phosphorylated4E-BP1 immunostaining was performed on 30 μm sections with a rabbit anti-phospho-4E-BP1 (Thr37/46) antibody (Cell Signaling, Beverly, MA) at 1:200. Sections were treated with biotinylated protein A and avidin-biotinylated horseradish peroxidase complexes (ABC; Vector Labs). After immunoperoxidase staining, sections were thionin counterstained. Quantitative morphologic analysis. To determine the number of

TH-positive neurons in the SN, both sides of each mouse brain were analyzed as described previously.17,37 The entire SN was identified as the region

www.moleculartherapy.org vol. 20 no. 2 feb. 2012

© The American Society of Gene & Cell Therapy

of interest by use of the StereoInvestigator program (MicroBrightField, Williston, VT). A fractionator probe was established for each section. The number of TH-positive neurons in each counting frame was determined by focusing down through the section, using ×100 objective under oil, as required by the optical disector method. Our criterion for counting an individual TH-positive neuron was the presence of its nucleus either within the counting frame, or touching the right or top frame lines (green), but not touching the left or bottom lines (red). The total number of TH-positive neurons for each side of the SN was then determined by the StereoInvestigator program. The size of TH-positive neurons in the SNpc and NeuN-positive neurons in the SN reticulata were also determined by use of the StereoInvestigator program. For each brain, four representative sections (one caudal, two middle, and one rostral) were chosen, and five neurons in each section were selected at random sites to provide 20 neurons per mouse brain. The area of each neuron was determined under ×100 oil-immersion objective. The optical density of striatal TH- and DAT-positive fibers was determined with an Imaging Research Analytical Imaging Station. The region of whole striatum was determined as previously described.17,37 TH- and DAT-positive fiber innervation of the striatum were expressed quantitatively as a percentage of optical density on the ipsilateral lesioned side compared with the contralateral control side. Measurement of dopamine and its metabolites. For determination of SN

and striatal levels of dopamine and its metabolites, each brain was placed in a mouse brain matrix and 2.0-mm thick coronal sections through the forebrain and mesencephalon were taken and the sections were placed flat on a chilled glass plate. The striatum was dissected on each side with a 2.0-mm tissue punch. The ventral mesencephalon (containing the SN) was dissected by a horizontal cut just dorsal to the SN. Tissues were frozen immediately on dry ice. Dopamine, 3,4-dihydroxyphenylacetic acid, and HVA were determined by high-performance liquid chromatography by Bioanalytical Systems (West Lafayette, IN ), and expressed as nanograms per sample.

Behavioral analysis. To assess rotational behavior, mice were injected with amphetamine (2.5 mg/kg intraperitoneal; Sigma) and placed free roaming in a plastic hemispherical bowl. They were allowed to habituate to their environment for 15 minutes, and then contralateral and ipsilateral turns were counted by a computerized rotometer system (San-Diego Instruments, San Diego, CA) for 60 minutes. Results were expressed as net turns per 60 minutes. Western analysis. For analysis of phospho-4E-BP1 (p-4E-BP1) expression,

each brain was placed in a mouse brain matrix and a 2.0-mm coronal slice through mesencephalon was taken for dissection of the SN, as described above, and lysed. Total protein concentration of the lysate was determined by microBCA kit (Pierce, Rockford, IL). Aliquots containing 30 µg of protein were electrophoresed in a SDS/polyacrylamide gel (Bio-Rad Labs, Hercules, CA) and transferred onto a Hybond-P membrane (Amersham Pharmacia Biosciences, Piscataway, NJ). The membrane was then probed with anti-phospho-4E-BP1 (Thr37/46) (Cell Signaling), anti-FLAG antibody (Sigma), or anti-β-actin and then incubated with appropriate secondary antibodies, conjugated to horseradish peroxidase, and detected with a chemiluminescent substrate (Pierce). Densitometric analysis of band intensity was performed by using a FluorChem 8800 Imaging System (Alpha Innotech, San Leandro, CA).

Statistical analysis. Differences between two groups were analyzed by the Student t-test. Multiple comparisons among groups were performed by one-way ANOVA and Tukey’s post hoc analysis. All statistical analyses were performed using SigmaStat software (Systat Software, San Leandro, CA).

ACKNOWLEDGMENTS This work was supported by NIH NS26836 and NS38370, the Parkinson’s disease Foundation, the Parkinson’s Alliance, and the RJG Foundation (R.E.B.).

Molecular Therapy vol. 20 no. 2 feb. 2012

Rheb Mediates Neuroprotection and Restoration

REFERENCES

1. Levi-Montalcini, R (1987). The nerve growth factor 35 years later. Science 237: 1154–1162. 2. Thoenen, H and Sendtner, M (2002). Neurotrophins: from enthusiastic expectations through sobering experiences to rational therapeutic approaches. Nat Neurosci 5 Suppl: 1046–1050. 3. Kirik, D, Georgievska, B and Björklund, A (2004). Localized striatal delivery of GDNF as a treatment for Parkinson disease. Nat Neurosci 7: 105–110. 4. Lin, LF, Doherty, DH, Lile, JD, Bektesh, S and Collins, F (1993). GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 260: 1130–1132. 5. Choi-Lundberg, DL, Lin, Q, Chang, YN, Chiang, YL, Hay, CM, Mohajeri, H et al. (1997). Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275: 838–841. 6. Choi-Lundberg, DL, Lin, Q, Chang, YN, Chiang, YL, Hay, CM, Mohajeri, H et al. (1997). Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275: 838–841. 7. Rosenblad, C, Martinez-Serrano, A and Björklund, A (1998). Intrastriatal glial cell linederived neurotrophic factor promotes sprouting of spared nigrostriatal dopaminergic afferents and induces recovery of function in a rat model of Parkinson’s disease. Neuroscience 82: 129–137. 8. Kordower, JH, Emborg, ME, Bloch, J, Ma, SY, Chu, Y, Leventhal, L et al. (2000). Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 290: 767–773. 9. Gill, SS, Patel, NK, Hotton, GR, O’Sullivan, K, McCarter, R, Bunnage, M et al. (2003). Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat Med 9: 589–595. 10. Lang, AE, Gill, S, Patel, NK, Lozano, A, Nutt, JG, Penn, R et al. (2006). Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease. Ann Neurol 59: 459–466. 11. Sherer, TB, Fiske, BK, Svendsen, CN, Lang, AE and Langston, JW (2006). Crossroads in GDNF therapy for Parkinson’s disease. Mov Disord 21: 136–141. 12. Airaksinen, MS and Saarma, M (2002). The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci 3: 383–394. 13. Pong, K, Xu, RY, Baron, WF, Louis, JC and Beck, KD (1998). Inhibition of phosphatidylinositol 3-kinase activity blocks cellular differentiation mediated by glial cell line-derived neurotrophic factor in dopaminergic neurons. J Neurochem 71: 1912–1919. 14. Soler, RM, Dolcet, X, Encinas, M, Egea, J, Bayascas, JR and Comella, JX (1999). Receptors of the glial cell line-derived neurotrophic factor family of neurotrophic factors signal cell survival through the phosphatidylinositol 3-kinase pathway in spinal cord motoneurons. J Neurosci 19: 9160–9169. 15. Encinas, M, Tansey, MG, Tsui-Pierchala, BA, Comella, JX, Milbrandt, J and Johnson, EM Jr (2001). c-Src is required for glial cell line-derived neurotrophic factor (GDNF) family ligand-mediated neuronal survival via a phosphatidylinositol-3 kinase (PI-3K)dependent pathway. J Neurosci 21: 1464–1472. 16. Ugarte, SD, Lin, E, Klann, E, Zigmond, MJ and Perez, RG (2003). Effects of GDNF on 6-OHDA-induced death in a dopaminergic cell line: modulation by inhibitors of PI3 kinase and MEK. J Neurosci Res 73: 105–112. 17. Ries, V, Henchcliffe, C, Kareva, T, Rzhetskaya, M, Bland, R, During, MJ et al. (2006). Oncoprotein Akt/PKB induces trophic effects in murine models of Parkinson’s disease. Proc Natl Acad Sci USA 103: 18757–18762. 18. Cheng, HC, Kim, SR, Oo, TF, Kareva, T, Yarygina, O, Rzhetskaya, M et al. (2011). Akt suppresses retrograde degeneration of dopaminergic axons by inhibition of macroautophagy. J Neurosci 31: 2125–2135. 19. Kaplitt, MG, Feigin, A, Tang, C, Fitzsimons, HL, Mattis, P, Lawlor, PA et al. (2007). Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet 369: 2097–2105. 20. Cheng, HC, Ulane, CM and Burke, RE (2010). Clinical progression in Parkinson disease and the neurobiology of axons. Ann Neurol 67: 715–725. 21. Choi, YJ, Di Nardo, A, Kramvis, I, Meikle, L, Kwiatkowski, DJ, Sahin, M et al. (2008). Tuberous sclerosis complex proteins control axon formation. Genes Dev 22: 2485–2495. 22. Campbell, DS and Holt, CE (2001). Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 32: 1013–1026. 23. Read, DE and Gorman, AM (2009). Involvement of Akt in neurite outgrowth. Cell Mol Life Sci 66: 2975–2984. 24. Park, KK, Liu, K, Hu, Y, Kanter, JL and He, Z (2010). PTEN/mTOR and axon regeneration. Exp Neurol 223: 45–50. 25. Avruch, J, Long, X, Lin, Y, Ortiz-Vega, S, Rapley, J, Papageorgiou, A et al. (2009). Activation of mTORC1 in two steps: Rheb-GTP activation of catalytic function and increased binding of substrates to raptor. Biochem Soc Trans 37(Pt 1): 223–226. 26. Urano, J, Comiso, MJ, Guo, L, Aspuria, PJ, Deniskin, R, Tabancay, AP Jr et al. (2005). Identification of novel single amino acid changes that result in hyperactivation of the unique GTPase, Rheb, in fission yeast. Mol Microbiol 58: 1074–1086. 27. Yan, L, Findlay, GM, Jones, R, Procter, J, Cao, Y and Lamb, RF (2006). Hyperactivation of mammalian target of rapamycin (mTOR) signaling by a gain-of-function mutant of the Rheb GTPase. J Biol Chem 281: 19793–19797. 28. Sato, T, Umetsu, A and Tamanoi, F (2008). Characterization of the Rheb-mTOR signaling pathway in mammalian cells: constitutive active mutants of Rheb and mTOR. Meth Enzymol 438: 307–320. 29. Urano, J, Sato, T, Matsuo, T, Otsubo, Y, Yamamoto, M and Tamanoi, F (2007). Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrientindependent mammalian TOR signaling in mammalian cells. Proc Natl Acad Sci USA 104: 3514–3519. 30. Sauer, H and Oertel, WH (1994). Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59: 401–415.

285

Rheb Mediates Neuroprotection and Restoration

31. Ries, V, Silva, RM, Oo, TF, Cheng, HC, Rzhetskaya, M, Kholodilov, N et al. (2008). JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra, but are not required for axon degeneration. J Neurochem 107: 1578–1588. 32. Kim, SR, Chen, X, Oo, TF, Kareva, T, Yarygina, O, Wang Cet al. (2011). Dopaminergic pathway reconstruction by Akt/Rheb-induced axon regeneration. Ann Neurol 70: 110–120. 33. Fearnley, JM and Lees, AJ (1991). Ageing and Parkinson’s disease: substantia nigra regional selectivity. Brain 114 (Pt 5): 2283–2301. 34. Pycock, CJ (1980). Turning behaviour in animals. Neuroscience 5: 461–514. 35. Silva, RM, Ries, V, Oo, TF, Yarygina, O, Jackson-Lewis, V, Ryu, EJ et al. (2005). CHOP/ GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism. J Neurochem 95: 974–986. 36. Martí, MJ, Saura, J, Burke, RE, Jackson-Lewis, V, Jiménez, A, Bonastre, M et al. (2002). Striatal 6-hydroxydopamine induces apoptosis of nigral neurons in the adult rat. Brain Res 958: 185–191. 37. Chen, X, Rzhetskaya, M, Kareva, T, Bland, R, During, MJ, Tank, AW et al. (2008). Antiapoptotic and trophic effects of dominant-negative forms of dual leucine zipper kinase in dopamine neurons of the substantia nigra in vivo. J Neurosci 28: 672–680. 38. Zigmond, MJ, Abercrombie, ED, Berger, TW, Grace, AA and Stricker, EM (1990). Compensations after lesions of central dopaminergic neurons: some clinical and basic implications. Trends Neurosci 13: 290–296. 39. Martin, DE and Hall, MN (2005). The expanding TOR signaling network. Curr Opin Cell Biol 17: 158–166. 40. Dunlop, EA and Tee, AR (2009). Mammalian target of rapamycin complex 1: signalling inputs, substrates and feedback mechanisms. Cell Signal 21: 827–835.

286

© The American Society of Gene & Cell Therapy

41. Foster, KG and Fingar, DC (2010). Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem 285: 14071–14077. 42. Laplante, M and Sabatini, DM (2009). mTOR signaling at a glance. J Cell Sci 122(Pt 20): 3589–3594. 43. Sarbassov, DD, Guertin, DA, Ali, SM and Sabatini, DM (2005). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098–1101. 44. Huang, J and Manning, BD (2008). The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem J 412: 179–190. 45. Huang, J and Manning, BD (2009). A complex interplay between Akt, TSC2 and the two mTOR complexes. Biochem Soc Trans 37(Pt 1): 217–222. 46. Datta, SR, Brunet, A and Greenberg, ME (1999). Cellular survival: a play in three Akts. Genes Dev 13: 2905–2927. 47. Downward, J (2004). PI 3-kinase, Akt and cell survival. Semin Cell Dev Biol 15: 177–182. 48. Burke, RE (2008). Programmed cell death and new discoveries in the genetics of parkinsonism. J Neurochem 104: 875–890. 49. Li, S, Takasu, T, Perlman, DM, Peterson, MS, Burrichter, D, Avdulov, S et al. (2003). Translation factor eIF4E rescues cells from Myc-dependent apoptosis by inhibiting cytochrome c release. J Biol Chem 278: 3015–3022. 50. Przedborski, S, Levivier, M, Jiang, H, Ferreira, M, Jackson-Lewis, V, Donaldson, D et al. (1995). Dose-dependent lesions of the dopaminergic nigrostriatal pathway induced by intrastriatal injection of 6-hydroxydopamine. Neuroscience 67: 631–647. 51. Kholodilov, N, Yarygina, O, Oo, TF, Zhang, H, Sulzer, D, Dauer, W et al. (2004). Regulation of the development of mesencephalic dopaminergic systems by the selective expression of glial cell line-derived neurotrophic factor in their targets. J Neurosci 24: 3136–3146.

www.moleculartherapy.org vol. 20 no. 2 feb. 2012