Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders

Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of Parkinson's disease. Our results suggest that AAV-Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as Parkinson's disease.     

Apoptosis inducing factor mediates caspase-independent 1-methyl-4-phenylpyridinium toxicity in dopaminergic cells

Parkinson's disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP+ toxicity, several recent studies have challenged the role of caspase-dependent apoptosis in dopaminergic neurons. Using the midbrain-derived MN9D dopaminergic cell line, we found that MPP+ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over-expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase-9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase-independent forms of regulated cell death following its translocation to the nucleus. We found that MPP+ treatment elicited nuclear translocation of AIF accompanied by large-scale DNA fragmentation. To establish the role of AIF in MPP+ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large-scale DNA fragmentation and conferred significant protection against MPP+ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase-independent cell death in MPP+-treated dopaminergic neurons.     

Erythropoietin protects against 6-hydroxydopamine-induced dopaminergic cell death

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopaminergic neurons. In the present study, erythropoietin, a trophic factor that has both hematopoietic and neural protective characteristics, was investigated for its capacity to protect dopaminergic neurons in experimental Parkinson's disease. Using both the dopaminergic cell line, MN9D, and primary dopamine neurons, we show that erythropoietin (1-3 U/mL) is neuroprotective against the dopaminergic neurotoxin, 6-hydroxydopamine. Protection was mediated by the erythropoietin receptor, as neutralizing anti-erythropoietin receptor antibody abrogated the protection. Activation of Akt/protein kinase B (PKB), via the phosphoinositide 3-kinase pathway, is a critical mechanism in erythropoietin-induced protection, while activation of extracellular signal-regulated kinase (ERK)1/2 contributes only moderately. Indeed, transfection of constitutively active Akt/PKB into dopaminergic cells was sufficient to protect against cell death. Furthermore, erythropoietin diminished markers of apoptosis in MN9D cells, including caspase 9 and caspase 3 activation and internucleosomal DNA fragmentation, suggesting that erythropoietin interferes with the apoptosis-execution process. When erythropoietin was administered to mice unilaterally lesioned with 6-hydroxydopamine, it prevented the loss of nigral dopaminergic neurons and maintained striatal catecholamine levels for at least 8 weeks. Erythropoietin-treated mice also had significantly reduced behavioral asymmetries. These studies suggest that erythropoietin can be an effective neuroprotective agent for dopaminergic neurons, and may be useful in reversing behavioral deficits associated with Parkinson's disease.     

A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.     

Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?

Bax is a proapoptotic member of the Bcl-2 family of proteins. It is believed to exert its action primarily by facilitating the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, leading to caspase activation and cell death. Because alterations in mitochondrial respiratory function, caspase activation and cell death with morphologic features compatible with apoptosis have been observed post mortem in the brain of patients with Parkinson's disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinson's disease post-mortem brain and primary mesencephalic cell cultures treated with MPP(+). We found that Bax is expressed ubiquitously by dopaminergic (DA) neurons in post-mortem brain of normal and Parkinson's disease subjects as well as in vitro. Using an antibody to Bax inserted into the outer mitochondrial membrane as an index of Bax activation, no significant differences were observed between control and Parkinson's disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinson's disease subjects, the percentage of Bax-positive melanized SNpc neurons containing Lewy bodies, suggestive of DA neuronal suffering, was significantly higher than the overall percentage of Bax-positive neurons among melanized neurons. Furthermore, all melanized SNpc neurons in Parkinson's disease subjects with activated caspase-3 were also immunoreactive for Bax, suggesting that Bax anchored in the outer mitochondrial membrane of melanized SNpc neurons showing signs of neuronal suffering or apoptosis is increased compared with DA neurons that are apparently unaltered. Surprisingly, MPP(+) treatment of tyrosine hydroxylase (TH)-positive neurons in primary mesencephalic cultures did not cause redistribution of Bax, although cytochrome c was released from the mitochondria and nuclear condensation/fragmentation was induced. Taken together, these findings suggest that in the human pathology, Bax may be a cofactor in caspase activation, but our in vitro data fail to indicate a central role for Bax in apoptotic death of DA neurons in an experimental Parkinson's disease paradigm.     

Zebrafish Parla- and Parlb-deficiency affects dopaminergic neuron patterning and embryonic survival

Many genes associated with familial Parkinson's disease contribute to mitochondrial morphology and function. Some of these genes, for example, Pink1 and Parkin, are part of a common pathway. The presenilin-associated rhomboid-like (PARL) gene was recently linked to familial Parkinson's disease. The PARL gene product is found in the inner mitochondrial membrane and cleaves the optic atrophy 1 protein, involved in mitochondrial morphology and apoptosis. In Drosophila, the PARL-related rhomboid-7 gene acts upstream of pink1 and parkin. However, such a genetic relationship is still unknown in vertebrates. Here, we show that the zebrafish genome comprises two parl paralogs: parla and parlb. Morpholino-mediated loss of parla and/or parlb function resulted in mild neurodegeneration, as evidenced by a lower density of dopaminergic neurons. Patterning of dopaminergic neurons was also perturbed in the ventral diencephalon. Morphants exhibited extensive cell death throughout the entire body as well as increased larval mortality. The morphant phenotype could be rescued by injection of human PARL mRNA, but not catalytically inactive PARL, suggesting functional conservation between the human and zebrafish proteins. More importantly, the zebrafish pink1 mRNA as well as the human PINK1 mRNA, but not kinase-dead nor Parkinson's disease-linked mutant PINK1 mRNA, also rescued the morphant phenotype, providing evidence that Parl genes may function upstream of Pink1, as part of a conserved pathway in vertebrates.     

Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death

Establishment of a Parkinson's disease (PD) neuron model was attempted with mouse embryonic stem (ES) cells. ES cell lines over-expressing mouse nuclear receptor-related 1 (Nurr1), together with human wild-type and alanine 30 --> proline (A30P) and alanine 53 --> threonine (A53T) mutant alpha-synuclein were established and subjected to differentiation into dopaminergic neurons. The ES cell-derived dopaminergic neurons expressing wild-type or mutant alpha-synuclein exhibited the fundamental characteristics consistent with dopaminergic neurons in the substantia nigra. The ES cell-derived PD model neurons exhibited increased susceptibility to oxidative stress, proteasome inhibition, and mitochondrial inhibition. Cell viability of PD model neurons and the control neurons was similar until 28 days after differentiation. Nonetheless, after that time, PD model neurons gradually began to undergo neuronal death over the course of 1 month, showing cytoplasmic aggregate formation and an increase of insoluble alpha-synuclein protein. Such delayed neuronal death was observed in a mutant alpha-synuclein protein level-dependent manner, which was slightly inhibited by a c-jun N-terminal kinase inhibitor and a caspase inhibitor. Such cell death was not observed when the same ES cell lines were differentiated into oligodendrocytes. The ES cell-derived PD model neurons are considered as prospective candidates for a new prototype modelling PD that would allow better investigation of the underlying neurodegenerative pathophysiology.     

Temporally controlled modulation of FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and human pluripotent stem cells

Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson's disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. In this study, we systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. We show that a brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, we provide evidence that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells.     

ApoER2 and VLDLr Are Required for Mediating Reelin Signalling Pathway for Normal Migration and Positioning of Mesencephalic Dopaminergic Neurons

The migration of mesencephalic dopaminergic (mDA) neurons from the subventricular zone to their final positions in the substantia nigra compacta (SNc), ventral tegmental area (VTA), and retrorubral field (RRF) is controlled by signalling from neurotrophic factors, cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM). Reelin and the cytoplasmic adaptor protein Disabled-1 (Dab1) have been shown to play important roles in the migration and positioning of mDA neurons. Mice lacking Reelin and Dab1 both display phenotypes characterised by the failure of nigral mDA neurons to migrate properly. ApoER2 and VLDLr are receptors for Reelin signalling and are therefore part of the same signal transduction pathway as Dab1. Here we describe the roles of ApoER2 and VLDLr in the proper migration and positioning of mDA neurons in mice. Our results demonstrate that VLDLr- and ApoER2-mutant mice have both a reduction in and abnormal positioning of mDA neurons. This phenotype was more pronounced in VLDLr-mutant mice. Moreover, we provide evidence that ApoER2/VLDLr double-knockout mice show a phenotype comparable with the phenotypes observed for Reelin- and Dab1- mutant mice. Taken together, our results demonstrate that the Reelin receptors ApoER2 and VLDLr play essential roles in Reelin-mediated migration and positioning of mDA neurons.     

Gene co-expression network analysis for identifying genetic markers in Parkinson's disease - a three-way comparative approach

Parkinson's disease (PD) is a neurodegenerative disorder involving progressive deterioration of dopaminergic neurons. Although few genetic markers for familial PD are known, the etiology of sporadic PD remains poorly understood. Microarray data was analysed for induced pluripotent stem cells (iPSCs) derived from PD patients and mature neuronal cells (mDA) differentiated from these iPSCs. Combining expression and semantic similarity, a highly-correlated PD interactome was constructed that included interactions of established Parkinson's disease marker genes. A novel three-way comparative approach was employed, delineating topologically and functionally important genes. These genes showed involvement in pathways like Parkin-ubiquitin proteosomal system (UPS), immune associated biological processes and apoptosis. Of interest are three genes, eEF1A1, CASK, and PSMD6 that are linked to PARK2 activity in the cell and thereby form attractive candidate genes for understanding PD. Network biology approach delineated in this study can be applied to other neurodegenerative disorders for identification of important genetic regulators.     

Hsp27 inhibits 6-hydroxydopamine-induced cytochrome c release and apoptosis in PC12 cells

Cellular stress may stimulate cell survival pathways or cell death depending on its severity. 6-Hydroxydopamine (6-OHDA) is a neurotoxin that targets dopaminergic neurons that is often used to induce neuronal cell death in models of Parkinson's disease. Here we present evidence that 6-OHDA induces apoptosis in rat PC12 cells that involves release of cytochrome c and Smac/Diablo from mitochondria, caspase-3 activation, cleavage of PARP, and nuclear condensation. 6-OHDA also induced the heat shock response, leading to increased levels of Hsp25 and Hsp70. Increased Hsp25 expression was associated with cell survival. Prior heat shock or overexpression of Hsp27 (human homologue of Hsp25) delayed cytochrome c release, caspase activation, and reduced the level of apoptosis caused by 6-OHDA. We conclude that 6-OHDA induces a variety of responses in cultured PC12 cells ranging from cell survival to apoptosis, and that induction of stress proteins such as Hsp25 may protect cells from undergoing 6-OHDA-induced apoptosis.     

Caspase-1 and caspase-8 cleave and inactivate cellular parkin

Lesions in the parkin gene cause early onset Parkinson's disease by a loss of dopaminergic neurons, thus demonstrating a vital role for parkin in the survival of these neurons. Parkin is inactivated by caspase cleavage, and the major cleavage site is after Asp126. Caspases responsible for parkin cleavage were identified by several experimental paradigms. Transient coexpression of caspases and wild type parkin in HEK-293 cells identified caspase-1, -3, and -8 as efficient inducers of parkin cleavage whereas caspase-2, -7, -9, and -11 did not induce cleavage. A D126A parkin mutation abrogates cleavage induced by caspase-1 and -8, but not by caspase-3. In anti-Fas-treated Jurkat T cells, parkin cleavage was inhibited by caspase inhibitors hFlip and CrmA (but not by X-linked inhibitor of apoptosis (XIAP)), indicating that caspase-8 (but not caspase-3) is responsible for the parkin cleavage in this model. Moreover, induction of apoptosis in caspase-3-deficient MCF7 cells, either by caspase-1 or -8 overexpression or by tumor necrosis factor-alpha treatment, led to parkin cleavage. These results demonstrate that caspase-1 and -8 can directly cleave parkin and suggest that death receptor activation and inflammatory stress can cause loss of the ubiquitin ligase activity of parkin, thus causing accumulation of toxic parkin substrates and triggering dopaminergic cell death.