Edward A. Burton

Bioenergetics of neurons inhibit the translocation response of Parkin following rapid mitochondrial depolarization

Recent studies delineate a pathway involving familial Parkinsons disease (PD)-related proteins PINK1 and Parkin, in which PINK1-dependent mitochondrial accumulation of Parkin targets depolarized mitochondria towards degradation through mitophagy. The pathway has been primarily characterized in cells less dependent on mitochondria for energy production than neurons. Here we report that in neurons, unlike other cells, mitochondrial depolarization by carbonyl cyanide m-chlorophenyl hydrazone did not induce Parkin translocation to mitochondria or mitophagy. PINK1 overexpression increased basal Parkin accumulation on neuronal mitochondria, but did not sensitize them to depolarization-induced Parkin translocation. Our data suggest that bioenergetic differences between neurons and cultured cell lines contribute to these different responses. In HeLa cells utilizing usual glycolytic metabolism, mitochondrial depolarization robustly triggered Parkin-mitochondrial translocation, but this did not occur in HeLa cells forced into dependence on mitochondrial respiration. Declining ATP levels after mitochondrial depolarization correlated with the absence of induced Parkin-mitochondrial translocation in both HeLa cells and neurons. However, intervention allowing neurons to maintain ATP levels after mitochondrial depolarization only modestly increased Parkin recruitment to mitochondria, without evidence of increased mitophagy. These data suggest that changes in ATP levels are not the sole determinant of the different responses between neurons and other cell types, and imply that additional mechanisms regulate mitophagy in neurons. Since the Parkin-mitophagy pathway is heavily dependent on bioenergetic status, the unique metabolic properties of neurons likely influence the function of this pathway in the pathogenesis of PD.

α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease

The Parkinson’s disease–associated protein α-synuclein binds to the mitochondrial protein import receptor TOM20, which causes downstream impairment of mitochondrial function. α-Synuclein disrupts the mitochondrial protein import business α-Synuclein accumulation and mitochondrial dysfunction are central to the pathogenesis of most forms of Parkinson’s disease and appear to intersect, but how the two are related to each other has remained elusive. Now, Di Maio and colleagues report that specific forms of wild-type α-synuclein, such as oligomeric and dopamine-modified forms, but not the monomeric or fibrillar forms, bind with high affinity to the mitochondrial receptor TOM20. This results in impaired import of proteins required for mitochondrial function and leads to senescence of mitochondria, which show reduced respiration and increased production of reactive oxygen species. This study also highlights potential ways to prevent this deleterious interaction and its downstream consequences. α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson’s disease (PD), and the two appear to be related. Mitochondrial dysfunction leads to accumulation and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment, but the basis for this bidirectional interaction remains obscure. We now report that certain posttranslationally modified species of α-synuclein bind with high affinity to the TOM20 (translocase of the outer membrane 20) presequence receptor of the mitochondrial protein import machinery. This binding prevented the interaction of TOM20 with its co-receptor, TOM22, and impaired mitochondrial protein import. Consequently, there were deficient mitochondrial respiration, enhanced production of reactive oxygen species, and loss of mitochondrial membrane potential. Examination of postmortem brain tissue from PD patients revealed an aberrant α-synuclein–TOM20 interaction in nigrostriatal dopaminergic neurons that was associated with loss of imported mitochondrial proteins, thereby confirming this pathogenic process in the human disease. Modest knockdown of endogenous α-synuclein was sufficient to maintain mitochondrial protein import in an in vivo model of PD. Furthermore, in in vitro systems, overexpression of TOM20 or a mitochondrial targeting signal peptide had beneficial effects and preserved mitochondrial protein import. This study characterizes a pathogenic mechanism in PD, identifies toxic species of wild-type α-synuclein, and reveals potential new therapeutic strategies for neuroprotection.

Muscle and Neural Isoforms of Agrin Increase Utrophin Expression in Cultured Myotubes via a Transcriptional Regulatory Mechanism

Duchenne muscular dystrophy is a prevalent X-linked neuromuscular disease for which there is currently no cure. Recently, it was demonstrated in a transgenic mouse model that utrophin could functionally compensate for the lack of dystrophin and alleviate the muscle pathology (Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I., and Davies, K. E. (1996) Nature 384, 349–353). In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In a recent study, we showed that the nerve exerts a profound influence on utrophin gene expression and postulated that nerve-derived trophic factors mediate the local transcriptional activation of the utrophin gene within nuclei located in the postsynaptic sarcoplasm (Gramolini, A. O., Dennis, C. L., Tinsley, J. M., Robertson, G. S., Davies, K. E, Cartaud, J., and Jasmin, B. J. (1997)J. Biol. Chem. 272, 8117–8120). In the present study, we have therefore focused on the effect of agrin on utrophin expression in cultured C2 myotubes. In response to Torpedo-, muscle-, or nerve-derived agrin, we observed a significant 2-fold increase in utrophin mRNAs. By contrast, CGRP treatment failed to affect expression of utrophin transcripts. Western blotting experiments also revealed that the increase in utrophin mRNAs was accompanied by an increase in the levels of utrophin. To determine whether these changes were caused by parallel increases in the transcriptional activity of the utrophin gene, we transfected muscle cells with a 1.3-kilobase pair utrophin promoter-reporter (nlsLacZ) gene construct and treated them with agrin for 24–48 h. Under these conditions, both muscle- and nerve-derived agrin increased the activity of β-galactosidase, indicating that agrin treatment led, directly or indirectly, to the transcriptional activation of the utrophin gene. Furthermore, this increase in transcriptional activity in response to agrin resulted from a greater number of myonuclei expressing the 1.3-kilobase pair utrophin promoter-nlsLacZ construct. Deletion of 800 base pairs 5′ from this fragment decreased the basal levels of nlsLacZ expression and abolished the sensitivity of the utrophin promoter to exogenously applied agrin. In addition, site-directed mutagenesis of an N-box motif contained within this 800-base pair fragment demonstrated its essential contribution in this regulatory mechanism. Finally, direct gene transfer studies performed in vivo further revealed the importance of this DNA element for the synapse-specific expression of the utrophin gene along multinucleated muscle fibers. These data show that both muscle and neural isoforms of agrin can regulate expression of the utrophin gene and further indicate that agrin is not only involved in the mechanisms leading to the formation of clusters containing presynthesized synaptic molecules but that it can also participate in the local regulation of genes encoding synaptic proteins. Together, these observations are therefore relevant for our basic understanding of the events involved in the assembly and maintenance of the postsynaptic membrane domain of the neuromuscular junction and for the potential use of utrophin as a therapeutic strategy to counteract the effects of Duchenne muscular dystrophy.

Gene delivery using herpes simplex virus vectors.

Herpes simplex virus (HSV) is a neurotropic DNA virus with many favorable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans, enabling straightforward production of high-titre pure preparations of non-pathogenic vector. The genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime. Transduction with replication-defective vectors causes a latent-like infection in both neural and non-neural tissue; the vectors are non-pathogenic, unable to reactivate and persist long-term. The latency active promoter complex can be exploited in vector design to achieve long-term stable transgene expression in the nervous system. HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed preliminary steps to be taken towards targeting the tropism of HSV vectors. Using replication-defective HSV vectors, highly encouraging results have emerged from recent pre-clinical studies on models of neurological disease, including glioma, peripheral neuropathy, chronic pain and neurodegeneration. Consequently, HSV vectors encoding appropriate transgenes to tackle these pathogenic processes are poised to enter clinical trials.

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid- β peptide in vivo

Accumulation of insoluble aggregates of amyloid-β peptide (Aβ), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimers disease (AD). Consequently, downregulation of APP, or enhanced clearance of Aβ, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Aβ accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Aβ (HSV-neprilysin), substantially inhibited accumulation of Aβ. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APPSw), resulted in rapid Aβ accumulation. Co-inoculation of LV-APPSw with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Aβ accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Aβ accumulation in the brain, thus providing useful tools to clarify the role of Aβ in AD that may facilitate the development of novel therapies for this important disease.

Gene therapy progress and prospects: Parkinson's disease.

GeriatricResearch, Education and Clinical Center (GRECC), Pittsburgh VA Healthcare System, Pittsburgh, PA, USAGene Therapy (2003) 10, 1721–1727. doi:10.1038/sj.gt.3302116PD is an attractive target for central nervous system(CNS) gene therapy for several reasons. First, thepathology in early PD is, to a first approximation, limitedto dopaminergic neurons projecting from the substantianigra pars compacta (SNc) to the caudate aputamenl, sothat localized gene delivery is a viable therapeuticstrategy. Second, the neurochemical deficits and thefunctional consequences of dopaminergic cell loss onlocal basal ganglia circuitry are well characterized; genetransfer can be designed either to improve cell survival,or to modify functional activity in the damaged basalganglia circuitry (summarized in Figures 1 and 2). Third,PD is common and disabling despite treatment; nocurrent intervention is uniformly accepted as altering thenatural history of disease progression; hence, develop-ment of novel therapeutics is desirable. A variety oftherapeutic transgenes has been delivered in experi-mental models of PD, using a number of differentvectors. In this article, we survey the literature from2000 to 2003, and briefly review recent progress in thedevelopment of gene transfer strategies for treating PD.

Generation of a transgenic zebrafish model of Tauopathy using a novel promoter element derived from the zebrafish eno2 gene

The aim of this study was to isolate cis-acting regulatory elements for the generation of transgenic zebrafish models of neurodegeneration. Zebrafish enolase-2 (eno2) showed neuronal expression increasing from 24 to 72 h post-fertilization (hpf) and persisting through adulthood. A 12 kb eno2 genomic fragment, extending from 8 kb upstream of exon 1 to exon 2, encompassing intron 1, was sufficient to drive neuronal reporter gene expression in vivo over a similar time course. Five independent lines of stable Tg(eno2 : GFP) zebrafish expressed GFP widely in neurons, including populations with relevance to neurodegeneration, such as cholinergic neurons, dopaminergic neurons and cerebellar Purkinje cells. We replaced the exon 2-GFP fusion gene with a cDNA encoding the 4-repeat isoform of the human microtubule-associated protein Tau. The first intron of eno2 was spliced with high fidelity and efficiency from the chimeric eno2-Tau transcript. Tau was expressed at ∼8-fold higher levels in Tg(eno2 : Tau) zebrafish brain than normal human brain, and localized to axons, neuropil and ectopic neuronal somatic accumulations resembling neurofibrillary tangles. The 12 kb eno2 promoter drives high-level transgene expression in differentiated neurons throughout the CNS of stable transgenic zebrafish. This regulatory element will be useful for the construction of transgenic zebrafish models of neurodegeneration.

Transgenic zebrafish models of neurodegenerative diseases

Since the introduction of the zebrafish as a model for the study of vertebrate developmental biology, an extensive array of techniques for its experimental manipulation and analysis has been developed. Recently it has become apparent that these powerful methodologies might be deployed in order to elucidate the pathogenesis of human neurodegenerative diseases and to identify candidate therapeutic approaches. In this article, we consider evidence that the zebrafish central nervous system provides an appropriate setting in which to model human neurological disease and we review techniques and resources available for generating transgenic models. We then examine recent publications showing that appropriate phenotypes can be provoked in the zebrafish through transgenic manipulations analogous to genetic abnormalities known to cause human tauopathies, polyglutamine diseases or motor neuron degenerations. These studies show proof of concept that findings in zebrafish models can be applicable to the pathogenic mechanisms underlying human diseases. Consequently, the prospects for providing novel insights into neurodegenerative diseases by exploiting transgenic zebrafish models and discovery-driven approaches seem favorable.

HSV trafficking and development of gene therapy vectors with applications in the nervous system

Herpes simplex virus type 1 (HSV-1) is a neurotropic double-stranded DNA virus that causes cold sores, keratitis, and rarely encephalitis in humans. Nonpathogenic HSV-1 gene transfer vectors have been generated by elimination of viral functions necessary for replication. The life cycle of the native virus includes replication in epithelial cells at the site of initial inoculation followed by retrograde axonal transport to the nuclei of sensory neurons innervating the area of cutaneous primary infection. In this review, we summarize the current understanding of the molecular basis for HSV cell entry, nuclear transport of the genome, virion egress following replication, and retrograde and anterograde axonal transport in neurons. We discuss how each of these properties has been exploited or modified to allow the generation of gene transfer vectors with particular utility for neurological applications. Recent advances in engineering virus entry have provided proof of principle that vector targeting is possible. Furthermore, significant and potentially therapeutic modifications to the pathological responses to various noxious insults have been demonstrated in models of peripheral nerve disease. These applications exploit the natural axonal transport mechanism of HSV, allowing transgene expression in the cell nucleus within the inaccessible trigeminal ganglion or dorsal root ganglion, following the noninvasive procedure of subcutaneous vector inoculation. These findings demonstrate the importance of understanding basic virology in the design of vector systems and the powerful approach of exploiting favorable properties of the parent virus in the generation of gene transfer vectors.

Genetic zebrafish models of neurodegenerative diseases

As a consequence of the widespread use of zebrafish in developmental biology studies, an extensive array of experimental tools and techniques has been assembled; it has recently become apparent that these might be exploited in the analysis of human neurodegenerative diseases. A surprising degree of functional conservation has been demonstrated between human genes implicated in neurodegenerative diseases and their zebrafish orthologues. In zebrafish models of recessive parkinsonism, Parkin or Pink1 knockdown gave rise to specific loss of dopamine neurons; in a zebrafish model of recessive spinal muscular atrophy, loss of Smn1 function caused specific motor axonal defects. In addition, pathological features of several dominant diseases were replicated by transgenic over-expression of mutant human proteins, including Tau, Huntingtin, and SOD1. In some cases, conservation of relevant cellular pathways was sufficient that disease-specific posttranslational changes to the respective proteins were found in the zebrafish models. These data collectively suggest that the zebrafish can be an appropriate setting in which to model the molecular events underlying human neuropsychiatric disease. Consequently, novel findings yielded by studies in zebrafish models may be applicable to human diseases; this is an exciting prospect, in view of the many potential uses of zebrafish models, for example, screening for lead therapeutic compounds, rapid functional assessments of putative modifier genes, and live observation of pathogenic mechanisms in vivo.