Combining Constitutively Active Rheb Expression and Chondroitinase Promotes Functional Axonal Regeneration after Cervical Spinal Cord Injury.

After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI.

Expressing Constitutively Active Rheb in Adult Neurons after a Complete Spinal Cord Injury Enhances Axonal Regeneration beyond a Chondroitinase-Treated Glial Scar.

After a spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to: (1) the presence of inhibitory molecules, e.g., chondroitin sulfate proteoglycans (CSPG), in the glial scar at the lesion; and (2) the diminished growth capacity of adult neurons. We sought to determine whether expressing a constitutively active form of the GTPase Rheb (caRheb) in adult neurons after a complete SCI in rats improves intrinsic growth potential to result in axon regeneration out of a growth-supportive peripheral nerve grafted (PNG) into the SCI cavity. We also hypothesized that treating the glial scar with chondroitinase ABC (ChABC), which digests CSPG, would further allow caRheb-transduced neurons to extend axons across the distal graft interface. We found that targeting this pathway at a clinically relevant post-SCI time point improves both sprouting and regeneration of axons. CaRheb increased the number of axons, but not the number of neurons, that projected into the PNG, indicative of augmented sprouting. We also saw that caRheb enhanced sprouting far rostral to the injury. CaRheb not only increased growth rostral and into the graft, it also resulted in significantly more regrowth of axons across a ChABC-treated scar into caudal spinal cord. CaRheb(+) neurons had higher levels of growth-associated-43, suggestive of a newly identified mechanism for mTOR-mediated enhancement of regeneration. Thus, we demonstrate for the first time that simultaneously addressing intrinsic and scar-associated, extrinsic impediments to regeneration results in significant regrowth beyond an extremely challenging, complete SCI site. SIGNIFICANCE STATEMENT: After spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to the diminished growth capacity of adult neurons and the presence of inhibitory molecules in the scar at the lesion. We sought to simultaneously counter both of these obstacles to achieve more robust regeneration after complete SCI. We transduced neurons postinjury to express a constitutively active Rheb to enhance their intrinsic growth potential, transplanted a growth supporting peripheral nerve graft into the lesion cavity, and enzymatically modulated the inhibitory glial scar distal to the graft. We demonstrate, for the first time, that simultaneously addressing neuron-related, intrinsic deficits in axon regrowth and extrinsic, scar-associated impediments to regeneration results in significant regeneration after SCI.

In vivo AAV1 transduction with hRheb(S16H) protects hippocampal neurons by BDNF production.

Recent evidence has shown that Ras homolog enriched in brain (Rheb) is dysregulated in Alzheimer's disease (AD) brains. However, it is still unclear whether Rheb activation contributes to the survival and protection of hippocampal neurons in the adult brain. To assess the effects of active Rheb in hippocampal neurons in vivo, we transfected neurons in the cornu ammonis 1 (CA1) region in normal adult rats with an adeno-associated virus containing the constitutively active human Rheb (hRheb(S16H)) and evaluated the effects on thrombin-induced neurotoxicity. Transduction with hRheb(S16H) significantly induced neurotrophic effects in hippocampal neurons through activation of mammalian target of rapamycin complex 1 (mTORC1) without side effects such as long-term potentiation impairment and seizures from the alteration of cytoarchitecture, and the expression of hRheb(S16H) prevented thrombin-induced neurodegeneration in vivo, an effect that was diminished by treatment with specific neutralizing antibodies against brain-derived neurotrophic factor (BDNF). In addition, our results showed that the basal mTORC1 activity might be insufficient to mediate the level of BDNF expression, but hRheb(S16H)-activated mTORC1 stimulated BDNF production in hippocampal neurons. These results suggest that viral vector transduction with hRheb(S16H) may have therapeutic value in the treatment of neurodegenerative diseases such as AD.

Rheb GTPase regulates ß-secretase levels and amyloid ß generation.

The ß-site amyloid precursor protein (APP)-cleaving enzyme 1 (ß-secretase, BACE1) initiates amyloidogenic processing of APP to generate amyloid ß (Aß), which is a hallmark of Alzheimer disease (AD) pathology. Cerebral levels of BACE1 are elevated in individuals with AD, but the molecular mechanisms are not completely understood. We demonstrate that Rheb GTPase (Ras homolog enriched in brain), which induces mammalian target of rapamycin (mTOR) activity, is a physiological regulator of BACE1 stability and activity. Rheb overexpression depletes BACE1 protein levels and reduces Aß generation, whereas the RNAi knockdown of endogenous Rheb promotes BACE1 accumulation, and this effect by Rheb is independent of its mTOR signaling. Moreover, GTP-bound Rheb interacts with BACE1 and degrades it through proteasomal and lysosomal pathways. Finally, we demonstrate that Rheb levels are down-regulated in the AD brain, which is consistent with an increased BACE1 expression. Altogether, our study defines Rheb as a novel physiological regulator of BACE1 levels and Aß generation, and the Rheb-BACE1 circuitry may have a role in brain biology and disease.

An early axonopathy in a hLRRK2(R1441G) transgenic model of Parkinson disease.

Mutations in the gene for LRRK2 are the most common cause of familial Parkinson's disease (PD) and patients with these mutations manifest clinical features that are indistinguishable from those of the more common sporadic form. Thus, investigations of disease mechanisms based on disease-causing LRRK2 mutations can be expected to shed light on the more common sporadic form as well as the inherited form. We have shown that as human BAC transgenic hLRRK2(R1441G) mice age, they exhibit two abnormalities in the nigrostriatal dopaminergic system: an axonopathy and a diminished number of dendrites in the substantia nigra (SN). To better understand disease mechanisms it is useful to determine where in the affected neural system the pathology first begins. We therefore examined the nigrostriatal dopaminergic system in young mice to determine the initial site of pathology. Brains from hLRRK2(R1441G) and littermate control mice at 2-4months of age were examined by immunohistochemistry, anterograde fluorescent axon labeling and ultrastructural analysis. SN neurons, their projecting axons and the striatal terminal fields were assessed. The first identifiable abnormality in this system is an axonopathy characterized by giant polymorphic axon spheroids, the presence of intra-axonal autophagic vacuoles and intra-axonal myelin invagination. An initial involvement of axons has also been reported for other genetic models of PD. These observations support the concept that axons are involved early in the course of the disease. We suggest that effective neuroprotective approaches will be aimed at preventing axonal degeneration.

Neurotrophic effects of serum- and glucocorticoid-inducible kinase on adult murine mesencephalic dopamine neurons.

Mesencephalic dopamine neurons are central to many aspects of human cognition, motivational, and motor behavior, and they are uniquely vulnerable to degenerative neurologic disorders such as Parkinson's disease. There is growing evidence that in the mature brain these neurons not only remain responsive to neurotrophic support, but are dependent on it for viability and function. Little is known of the cellular signaling pathways that mediate this support, although some evidence suggests that protein kinase Akt/PKB may play such a role. Another candidate for such a role is serum- and glucocorticoid-inducible kinase (SGK), a member of the AGC kinase family that is closely related to Akt. We have herein examined the responsiveness of adult mouse dopamine neurons in vivo to overexpression of wild-type and a constitutively active form of SGK by use of viral vector transfer in normal mice and both before and after 6-OHDA lesion. We find that SGK induces a broad spectrum of neurotrophic effects on these neurons, including induction of neuronal hypertrophy, protection from both neuron death and neurotoxin-induced retrograde axonal degeneration, and axon regeneration. Given the diverse and robust effects of SGK on these neurons, and its abundant expression in them, we suggest that SGK, like closely related Akt, may play a role in their responsiveness to neurotrophic factors and in adult maintenance. It therefore offers a novel target for therapeutic development.

AAV transduction of dopamine neurons with constitutively active Rheb protects from neurodegeneration and mediates axon regrowth.

There are currently no therapies that provide either protection or restoration of neuronal function for adult-onset 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.

Akt suppresses retrograde degeneration of dopaminergic axons by inhibition of macroautophagy.

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.

Dopaminergic pathway reconstruction by Akt/Rheb-induced axon regeneration.

OBJECTIVE: A prevailing concept in neuroscience has been that the adult mammalian central nervous system is incapable of restorative axon regeneration. Recent evidence, however, has suggested that reactivation of intrinsic cellular programs regulated by protein kinase B (Akt)/mammalian target of rapamycin (mTor) signaling may restore this ability. METHODS: To assess this possibility in the brain, we have examined the ability of adenoassociated virus (AAV)-mediated transduction of dopaminergic neurons of the substantia nigra (SN) with constitutively active forms of the kinase Akt and the GTPase Ras homolog enriched in brain (Rheb) to induce regrowth of axons after they have been destroyed by neurotoxin lesion. RESULTS: Both constitutively active myristoylated Akt and hRheb(S16H) induce regrowth of axons from dopaminergic neurons to their target, the striatum. Histological analysis demonstrates that these new axons achieve morphologically accurate reinnervation. In addition, functional reintegration into target circuitry is achieved, as indicated by partial behavioral recovery. INTERPRETATION: We conclude that regrowth of axons within the adult nigrostriatal projection, a system that is prominently affected in Parkinson's disease, can be achieved by activation of Akt/mTor signaling in surviving endogenous mesencephalic dopaminergic neurons by viral vector transduction.

Glial cell line-derived neurotrophic factor receptor-α1 expressed in striatum in trans regulates development and injury response of dopamine neurons of the substantia nigra.

Many of the cellular effects of glial cell line-derived neurotrophic factor are initiated by binding to GNDF family receptor alpha-1 (GFRα1), and mediated by diverse intracellular signaling pathways, most notably through the Ret tyrosine kinase. Ret may be activated by the cell autonomous expression of GFRα1 ('in cis'), or by its non-cell autonomous presence ('in trans'), in either a soluble or immobilized state. GFRα1 is expressed in the striatum, a target of the dopaminergic projection of the substantia nigra. To determine whether post-synaptic expression of GFRα1 in striatum in trans has effects on the development or adult responses to injury of dopamine neurons, we have created transgenic mice in which GFRα1 expression is selectively increased in striatum and other forebrain targets of the dopaminergic projection. Post-synaptic GFRα1 has profound effects on the development of dopamine neurons, resulting in a 40% increase in their adult number. This morphologic effect was associated with an augmented motor response to amphetamine. In adult mice, post-synaptic GFRα1 expression did not affect neuron survival following neurotoxic lesion, but it did increase the preservation of striatal dopaminergic innervation. We conclude that post-synaptic striatal GFRα1 expression has important effects on the biology of dopamine neurons in vivo.

Age and α-synuclein expression interact to reveal a dependence of dopaminergic axons on endogenous Akt/PKB signaling.

The mechanisms underlying the chronic neurodegeneration that occurs in Parkinson's disease (PD) are unknown. One emerging hypothesis is that neural systems deteriorate and eventually degenerate due to a primary failure of either extrinsic neurotrophic support or the intrinsic cellular pathways that mediate such support. One of the cellular pathways that have been often identified in mediating neurotrophic effects is that of PI3K/Akt signaling. In addition, recent observations have suggested a primary failure of PI3K/Akt signaling in animal models and in PD patients. Therefore, to explore the possible role of endogenous Akt signaling in maintaining the viability and functionality of substantia nigra (SN) dopamine neurons, one of the principal systems affected in PD, we have used an adeno-associated viral vector to transduce them with a dominant negative (DN) form of Akt, the pleckstrin homology (PH) domain alone (DN(PH)-Akt). In addition, we have examined the effect of DN(PH)-Akt in murine models of two risk factors for human PD: advanced age and increased expression of α-synuclein. We find that transduction of these neurons in normal adult mice has no effect on any aspect of their morphology at 4 or 7weeks. However, in both aged mice and in transgenic mice with increased expression of human α-synuclein we observe decreased phenotypic expression of the catecholamine synthetic enzyme tyrosine hydroxylase (TH) in dopaminergic axons and terminals in the striatum. In aged transgenic α-synuclein over-expressing mice this reduction was 2-fold as great. We conclude that the two principal risk factors for human PD, advanced age and increased expression of α-synuclein, reveal a dependence of dopaminergic neurons on endogenous Akt signaling for maintenance of axonal phenotype.

Brain-derived neurotrophic factor regulates early postnatal developmental cell death of dopamine neurons of the substantia nigra in vivo.

Brain-derived neurotrophic factor (BDNF) was the first purified molecule identified to directly support the development of mesencephalic dopamine neurons. However, its physiologic role has remained unknown. Based on patterns of expression, it is unlikely to serve as a target-derived neurotrophic factor, but it may instead act locally in the mesencephalon, either released by afferent projections, or in autocrine fashion. To assess a possible local role, we blocked BDNF signaling in the substantia nigra (SN) of postnatal rats by injection of either neutralizing antibodies or a peptide antagonist. These treatments increased the magnitude of developmental cell death in the SN, indicating that endogenous local BDNF does play a regulatory role. However, we also find that elimination of BDNF in brain throughout postnatal development in BDNF(fl/fl):Nestin-Cre mice has no effect on the adult number of SN dopamine neurons. We postulate that other forms of trophic support may compensate for the elimination of BDNF during early development. Although the number of SN dopamine neurons is unchanged, their organization is disrupted. We conclude that BDNF plays a physiologic role in the postnatal development of SN dopamine neurons.

Antiapoptotic and trophic effects of dominant-negative forms of dual leucine zipper kinase in dopamine neurons of the substantia nigra in vivo.

There is extensive evidence that the mitogen-activated protein kinase (MAPK) signaling cascade mediates programmed cell death in neurons. However, current evidence that the mixed linage kinases (MLKs), upstream in this cascade, mediate cell death is based, in the in vivo context, entirely on pharmacological approaches. The compounds used in these studies have neither complete specificity nor selectivity among these kinases. Therefore, to better address the molecular specificity of the MLKs in mediating neuron death, we used dominant-negative constructs delivered by AAV (adenoassociated virus) vector transfer. We assessed effects in a neurotoxin model of parkinsonism, in which neuroprotection by pharmacologic MLK inhibition has been reported. We find that two dominant-negative forms of dual leucine zipper kinase (DLK) inhibit apoptosis and enhance long-term survival of dopamine neurons, but a dominant negative of MLK3 does not. Interestingly, the kinase-dead form of DLK not only blocks apoptosis but also has trophic effects on dopamine neurons. Although the MAPK cascade activates a number of downstream cell death mediators, we find that inhibition of DLK correlates closely with blockade of phosphorylation of c-jun and prevention of cell death. We conclude that DLK acts primarily through c-jun phosphorylation to mediate cell death in this model.

Regulation of the postnatal development of dopamine neurons of the substantia nigra in vivo by Akt/protein kinase B.

Following mitosis, specification and migration during embryogenesis, dopamine neurons of the mesencephalon undergo a postnatal naturally occurring cell death event that determines their final adult number, and a period of axonal growth that determines pattern and extent of target contacts. While a number of neurotrophic factors have been suggested to regulate these developmental events, little is known, especially in vivo, of the cell signaling pathways that mediate these effects. We have examined the possible role of Akt/Protein Kinase B by transduction of these neurons in vivo with adeno-associated viral vectors to express either a constitutively active or a dominant negative form of Akt/protein kinase B. We find that Akt regulates multiple features of the postnatal development of these neurons, including the magnitude of the apoptotic developmental cell death event, neuron size, and the extent of target innervation of the striatum. Given the diversity and magnitude of its effects, the regulation of the development of these neurons by Akt may have implications for the many psychiatric and neurologic diseases in which these neurons may play a role.

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra, but are not required for axon degeneration.

Activation of c-jun N-terminal kinase (JNK) by the mitogen-activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson's disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6-hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.

Context-dependent expression of a conditionally-inducible form of active Akt.

Akt kinases are key signaling components in proliferation-competent and post-mitotic cells. Here, we sought to create a conditionally-inducible form of active Akt for both in vitro and in vivo applications. We fused a ligand-responsive Destabilizing Domain (DD) derived from E. coli dihydrofolate reductase to a constitutively active mutant form of Akt1, Akt(E40K). Prior work indicated that such fusion proteins may be stabilized and induced by a ligand, the antibiotic Trimethoprim (TMP). We observed dose-dependent, reversible induction of both total and phosphorylated/active DD-Akt(E40K) by TMP across several cellular backgrounds in culture, including neurons. Phosphorylation of FoxO4, an Akt substrate, was significantly elevated after DD-Akt(E40K) induction, indicating the induced protein was functionally active. The induced Akt(E40K) protected cells from apoptosis evoked by serum deprivation and was neuroprotective in two cellular models of Parkinson's disease (6-OHDA and MPP+ exposure). There was no significant protection without induction. We also evaluated Akt(E40K) induction by TMP in mouse substantia nigra and striatum after neuronal delivery via an AAV1 adeno-associated viral vector. While there was significant induction in striatum, there was no apparent induction in substantia nigra. To explore the possible basis for this difference, we examined DD-Akt(E40K) induction in cultured ventral midbrain neurons. Both dopaminergic and non-dopaminergic neurons in the cultures showed DD-Akt(E40K) induction after TMP treatment. However, basal DD-Akt(E40K) expression was 3-fold higher for dopaminergic neurons, resulting in a significantly lower induction by TMP in this population. Such findings suggest that dopaminergic neurons may be relatively inefficient in protein degradation, a property that could relate to their lack of apparent DD-Akt(E40K) induction in vivo and to their selective vulnerability in Parkinson's disease. In summary, we generated an inducible, biologically active form of Akt. The degree of inducibility appears to reflect cellular context that will inform the most appropriate applications for this and related reagents.

Protection of nigral dopaminergic neurons by AAV1 transduction with Rheb(S16H) against neurotoxic inflammation in vivo.

We recently reported that adeno-associated virus serotype 1 (AAV1) transduction of murine nigral dopaminergic (DA) neurons with constitutively active ras homolog enriched in brain with a mutation of serine to histidine at position 16 [Rheb(S16H)] induced the production of neurotrophic factors, resulting in neuroprotective effects on the nigrostriatal DA system in animal models of Parkinson's disease (PD). To further investigate whether AAV1-Rheb(S16H) transduction has neuroprotective potential against neurotoxic inflammation, which is known to be a potential event related to PD pathogenesis, we examined the effects of Rheb(S16H) expression in nigral DA neurons under a neurotoxic inflammatory environment induced by the endogenous microglial activator prothrombin kringle-2 (pKr-2). Our observations showed that Rheb(S16H) transduction played a role in the neuroprotection of the nigrostriatal DA system against pKr-2-induced neurotoxic inflammation, even though there were similar levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1ß), in the AAV1-Rheb(S16H)-treated substantia nigra (SN) compared to the SN treated with pKr-2 alone; the neuroprotective effects may be mediated by the activation of neurotrophic signaling pathways following Rheb(S16H) transduction of nigral DA neurons. We conclude that AAV1-Rheb(S16H) transduction of neuronal populations to activate the production of neurotrophic factors and intracellular neurotrophic signaling pathways may offer promise for protecting adult neurons from extracellular neurotoxic inflammation.

Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo.

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson's disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes PD.

A quantitative evaluation of a 2.5-kb rat tyrosine hydroxylase promoter to target expression in ventral mesencephalic dopamine neurons in vivo.

Adeno-associated viruses (AAVs) have become powerful tools in neuroscience for both basic research and potential therapeutic use. They have become especially important tools for optogenetic experiments based on their ability to achieve transgene expression in postmitotic neurons with regional selectivity. With the use of appropriate promoter elements they can achieve cellular specificity as well. One population of neurons that plays a central role in human neurodegenerative and psychiatric diseases are the dopamine neurons of the midbrain. To study these neurons in vivo with advanced techniques it would be highly advantageous to characterize an appropriate specific promoter. To this end we have characterized a 2.5-kb sequence of the rat tyrosine hydroxylase (TH) promoter. The rTHp(2.5) promoter induced expression of the fluorescent reporter protein mCherry in SN dopamine neurons. Although it showed excellent specificity in cortex and striatum, where no reporter expression was observed, in the SN region many neurons expressed reporter but not TH. We show that some of the TH negativity is due to the suppression of its expression by the transgene. We conclude that rTHp(2.5) does preferentially label dopamine neurons but its specificity is not complete within the substantia nigra and caution must be used.

Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo.

The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.