The introduction of mutant, disease-causing genes into the germline of rodents and other animals has been a great boost to neurodegenerative disease research, but there are some limitations to the method, including the time and expense it takes to raise transgenic animals and the possibility that the introduced genes fundamentally alter development. One of the most promising alternatives is the introduction of transgenes into older animals via viruses, but early models were beset by problems with viral toxicity and efficient gene expression. Recent versions have overcome these limitations, and several groups have now used the recombinant adeno-associated virus (rAAV) vector to create or treat models of Parkinson's disease.

Killing Dopaminergic Cells with α-Synuclein
Two groups working independently--Ron Klein and colleagues at the University of Florida in Gainesville and Deniz Kirik, Anders Bjorklund and colleagues at Lund University in Sweden--have produced parkinsonian pathology by using rAAV vectors to overexpress α-synuclein in rats. Bjorklund's group was also able to show Parkinson-like behavioral deficits.

α-Synuclein is a major constituent of Lewy bodies and dystrophic neurites, both pathological hallmarks of PD. Mutations in the gene for α-synuclein can cause familial forms of the disease. Germline transgenic mouse models expressing these mutated genes have mimicked damage to axons and motor impairments, but have failed to produce the characteristic selective loss of dopaminergic cells in the substantia nigra (SN).

Klein's group, reporting in the 20 March Human Gene Therapy, used rAAV to express a mutant human α-synuclein gene. They showed accumulation of α-synuclein in SN neurons, Lewy-like dystrophic neurites in the SN and its target area, the striatum, and a 53 percent loss of dopaminergic neurons.

Bjorklund's group, reporting in the April 1 Journal of Neuroscience, expressed a different human mutant gene, as well as wildtype human α-synuclein. They demonstrated Lewy-like α-synuclein accumulation in the dopaminergic cells, as well as dystrophic neurites, accompanied by large-scale loss of dopaminergic cells and depletion of dopamine in the striatum. They also found significant motor impairment in animals whose cell loss exceeded 50-60 percent. Interestingly, both the human wildtype and mutant α-synuclein genes produced these effects.

Saving Dopaminergic Cells With L-Dopa
Kirik, Bjorklund and colleagues also demonstrate a more complex, therapeutic use of the rAAV vector approach in yesterday's Proceedings of the National Academy of Sciences (USA). They delivered a constant supply of the dopamine precursor levodopa (L-dopa) to the striatum in a mouse model of Parkinson's and thereby reduced, or eliminated, motor impairments.

The intermittent, pulsatile bioavailability of current oral L-dopa treatment in humans is thought to be partly responsible for the drug's diminishing effectiveness and increasing side effects when given for long periods of time. Viral vectors containing the gene for the enzyme tyrosine hydroxylase (TH), which synthesizes L-dopa, have been deployed to the striatum in an attempt to boost dopamine levels, alas without success. The Swedish group used the rAAV vector to deliver genes for TH and a needed cofactor to the striatum of animals with partial or total lesions of the nigrostriatal pathway.

Even in animals with total lesions, this restored striatal L-dopa production to levels sufficient to partially reverse the behavioral effects of the lesions. In animals where some of the nigrostriatal pathway was left intact (a state that perhaps more accurately reflects the situation in PD), the transgenes completely reversed the behavioral deficits of the lesions.

"These data suggest that local intrastriatal L-dopa delivery may be a viable therapeutic strategy in PD not only for treatment of underlying motor deficits but also for control of adverse side effects associated with oral L-dopa therapy such as on-off fluctuations and drug-induced dyskinesias," the authors conclude.-Hakon Heimer.

References:
Klein RL, King MA, Hamby ME, Meyer EM. Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra. Hum Gene Ther 2002 Mar 20;13(5):605-12. Abstract

Kirik D, Rosenblad C, Burger C, Lundberg C, Johansen TE, Muzyczka N, Mandel RJ, Bjorklund A. Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J Neurosci 2002 Apr 1;22(7):2780-91. Abstract

Kirik D, Georgievska B, Burger C, Winkler C, Muzyczka N, Mandel RJ, Bjorklund A. Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer. Proc Natl Acad Sci U S A 2002 Apr 2;99(7):4708-13. Abstract

Related ARF News Story, Related ARF News Story

Comments

Make a Comment

To make a comment you must login or register.

Comments on this content

  1. While the germline transgenic approach is the gold standard for studying gene function in the brain, it is also labor-intensive, expensive, and requires long periods of time to screen and raise the animals. We have adopted an alternative somatic transgenic strategy (historically applied for gene therapy) where the mutant genes can be expressed directly in the brains of adult animals, a method that by comparison, is inexpensive and fast.

    The application of a somatic-cell transgenic approach to modeling neurodegenerative diseases will be useful for several reasons, including: (1) temporal control of expression to avoid developmental effects and also to study aging effects, i.e. similar periods of expression in young or aged subjects; (2) spatial control of expression to target brain regions associated with specific disease states; and (3) transgene combinations.

    Further, the vector-based approaches can be applied to rats or monkeys and also take advantage of internal controls, i.e. comparisons to the contralateral, untreated hemisphere. Despite some real limitations of this approach, such as the volume of brain that can be affected and the size of the DNA that can be incorporated, we predict that somatic transgenic models of neurodegenerative diseases will be useful to study the disease process.

    Somatic gene transfer offers several specific advantages for neurodegenerative disease-modeling. The neurodegenerative diseases are typically associated with specific parts of the brain and are age-related. The spatio-temporal control of expression that is possible with the somatic gene transfer is therefore a powerful way to model these features of the disease process. Further, these diseases are often multi-factorial, and there is greater facility to combine genetic manipulations in the somatic transgenic approach compared to germ?line transgenic mice.

    It is unclear why the AAV vector system was so effective in our study, although it could be due to its ability to express high levels of the gene product selectively in this brain region, and perhaps also to the application to adults, avoiding some form of developmental compensation. Due to the complex nature of these models and hypotheses tested, the similar results obtained from Dr. Bjorklund and colleagues, a foremost laboratory in Parkinson's disease research, are an important confirmation of our work and the significance of the approach.

    We are currently studying other factors linked to Parkinson's disease that may block or exacerbate the cell loss, and hope that these approaches will help us understand why dopamine neurons are vulnerable during Parkinson's disease, as well as lead toward novel therapies, including gene therapy.

  2. I believe the most important aspect of our study is that we describe a new approach to study protein dysfunction in appropriate animal models with a strong basis in morphological, biochemical as well as behavioral assessments. Our model offers numerous advantages over transgenic mouse models. For example, it can easily be generated using wild-type animals in sufficient numbers with a very simple surgical intervention; the animals can be rendered transgenic at any time during their lifetime; it can be applied unilaterally leaving the contralateral side as an internal control; detailed functional assessments can better be done using rats as compared with mice; the model can be applied to primates to answer certain question that cannot be satisfactorily addressed in rodents.

    The method is very reproducible. We have now done (including new experiments not included in this paper) over 500 surgeries, and in all cases we can hit nearly all of the nigral dopamine cells with very high precision. This model is unique because the AAV vectors have a high affinity to these cells.

    As to the applicability of these methods to Alzheimer's research, to my knowledge there are no data with AD genes such as AβPP, presenilins, or tau. We are very interested in doing such work and have recently initiated efforts in this direction.

    The vectors Dr. Klein and colleagues used were prepared with essentially the same procedures as ours, except that they used the A30P mutant form of the human gene, whereas we used A53T mutation and the wild-type human genes. I should mention, however, that our interpretation of these data and some others (yet unpublished) is that the mutation does not seem to augment the pathology. Note also that wild-type human a-synuclein is different from the wild-type rat α-synuclein.

    Klein and colleagues report a similar cell loss of the TH+ cells in the substantia nigra as in our study, but their morphological analysis of the data is limited to this. No studies have been included to look at biochemical changes or the time course of the degeneration, and no tracing studies were performed. Documentation of α-synuclein-induced pathology is superficial; the descriptions of Lewy-like axonal pathologies are based solely on marker protein GFP.

    Klein and colleagues have concluded that, in their animals, they did not see behavioral impairments. This can easily be explained in two ways: First, the behavioral testing paradigms applied in their study are insufficient to pick up impairments in animals with mild-to-moderate degeneration in the ascending dopamine system. Second, they have tested a very small group of animals in their study. This dramatically reduces the power of their analysis.

    We observed that about 25 percent of the animals manifest behavioral impairments, while the majority seems to have compensated for their neuron loss. The impairments could, however, be revealed by use of sub-threshold blockade of TH enzyme in the seemingly normal animals. If we come back to the Klein experiment, it is natural for them to conclude that there was no apparent behavioral deficit in their animals as they have looked at only 6-8 of them. So, briefly, I believe that their data is essentially supporting and replicating a subset of ours, but the difference is that it has not been analyzed as carefully and with appropriate detail.

  3. Use of viral vector for targeting transgene expression in a region- and time-specific manner was first demonstrated to model a neurodegenerative disorder, namely trinucleotide repeat disease, by Fred Gage's lab (Senut et al., 2000). This approach offers a unique opportunity for the development and modeling of neurodegenerative disorders in a rapid and reliable manner. It also offers a unique opportunity of testing in vivo selective neuronal vulnerability and the differential effects of mutations.

    A similar approach is currently under development by our group in collaboration with Fred Gage and Inder Verma to model Alzheimer's disease and other neurodegenerative disorders using lentiviral vectors. This approach also holds great promise for the development of new treatments for PD.

Comments on Primary Papers for this Article

No Available Comments on Primary Papers for this Article

References

News Citations

  1. Adenovirus-Mediated Local Modification of Brain Genotype
  2. Transgenics Made Easy? Lentivirus Found to Carry Genes into Mice, Rats

Paper Citations

  1. . Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra. Hum Gene Ther. 2002 Mar 20;13(5):605-12. PubMed.
  2. . Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J Neurosci. 2002 Apr 1;22(7):2780-91. PubMed.
  3. . Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4708-13. PubMed.

Further Reading

Papers

  1. . Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra. Hum Gene Ther. 2002 Mar 20;13(5):605-12. PubMed.
  2. . Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J Neurosci. 2002 Apr 1;22(7):2780-91. PubMed.
  3. . Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4708-13. PubMed.

News

  1. Transgenics Made Easy? Lentivirus Found to Carry Genes into Mice, Rats
  2. Adenovirus-Mediated Local Modification of Brain Genotype

Primary Papers

  1. . Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra. Hum Gene Ther. 2002 Mar 20;13(5):605-12. PubMed.
  2. . Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system. J Neurosci. 2002 Apr 1;22(7):2780-91. PubMed.
  3. . Reversal of motor impairments in parkinsonian rats by continuous intrastriatal delivery of L-dopa using rAAV-mediated gene transfer. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4708-13. PubMed.