Jason R. Cannon

A highly reproducible rotenone model of Parkinson's disease

The systemic rotenone model of Parkinsons disease (PD) accurately replicates many aspects of the pathology of human PD and has provided insights into the pathogenesis of PD. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion and the extent of that lesion. The goal here was to develop an improved and highly reproducible rotenone model of PD. In these studies, male Lewis rats in three age groups (3, 7 or 12-14 months) were administered rotenone (2.75 or 3.0 mg/kg/day) in a specialized vehicle by daily intraperitoneal injection. All rotenone-treated animals developed bradykinesia, postural instability, and/or rigidity, which were reversed by apomorphine, consistent with a lesion of the nigrostriatal dopamine system. Animals were sacrificed when the PD phenotype became debilitating. Rotenone treatment caused a 45% loss of tyrosine hydroxylase-positive substantia nigra neurons and a commensurate loss of striatal dopamine. Additionally, in rotenone-treated animals, alpha-synuclein and poly-ubiquitin positive aggregates were observed in dopamine neurons of the substantia nigra. In summary, this version of the rotenone model is highly reproducible and may provide an excellent tool to test new neuroprotective strategies.

The Role of Environmental Exposures in Neurodegeneration and Neurodegenerative Diseases

Neurodegeneration describes the loss of neuronal structure and function. Numerous neurodegenerative diseases are associated with neurodegeneration. Many are rare and stem from purely genetic causes. However, the prevalence of major neurodegenerative diseases is increasing with improvements in treating major diseases such as cancers and cardiovascular diseases, resulting in an aging population. The neurological consequences of neurodegeneration in patients can have devastating effects on mental and physical functioning. The causes of most cases of prevalent neurodegenerative diseases are unknown. The role of neurotoxicant exposures in neurodegenerative disease has long been suspected, with much effort devoted to identifying causative agents. However, causative factors for a significant number of cases have yet to be identified. In this review, the role of environmental neurotoxicant exposures on neurodegeneration in selected major neurodegenerative diseases is discussed. Alzheimers disease, Parkinsons disease, multiple sclerosis, and amyotrophic lateral sclerosis were chosen because of available data on environmental influences. The special sensitivity the nervous system exhibits to toxicant exposure and unifying mechanisms of neurodegeneration are explored.

Chronic rotenone exposure reproduces Parkinson's disease gastrointestinal neuropathology

Gastrointestinal disorders, particularly severe constipation and delayed gastric emptying, are core symptoms of Parkinsons disease that affect most patients. However, the neuropathological substrate and physiological basis for this dysfunction are poorly defined. To begin to explore these phenomena in laboratory models of PD, rats were treated with either vehicle or rotenone (2.0 mg/kg, i.p.; 5 days/week) for 6-weeks. Myenteric plexus alpha-synuclein aggregate pathology and neuron loss were assessed 3-days and 6-months after the last rotenone injection. Gastrointestinal motility was assessed at 3-days, 1-month and 6-months after the last rotenone injection. Rotenone treatment caused an acute reduction in alpha-synuclein-immunoreactivity, but this was followed 6 months later by a robust increase in aggregate pathology and cytoplasmic inclusions that were similar in appearance to enteric Lewy-bodies in idiopathic PD. Rotenone-treated rats also had a moderate but permanent loss of small intestine myenteric neurons and an associated modest slowing of gastrointestinal motility 6-months after treatment. Our results suggest that a circumscribed exposure to an environmental toxicant can cause the delayed appearance of parkinsonian alpha-synuclein pathology in the enteric nervous system and an associated functional deficit in gastrointestinal motility. The rotenone model may therefore, provide a means to investigate pathogenic mechanisms and to test new therapeutic interventions into gastrointestinal dysfunction in PD.

Neurotoxic in vivo models of Parkinson’s disease: recent advances

Animal models have been invaluable to Parkinsons disease (PD) research. Of these, neurotoxin models have historically been the most widely utilized. The goal of this chapter is to give a brief historical description of classic PD models and then to identify the most recent important advances in modeling human PD in animals. Indeed, significant advances in modeling additional features of PD and expansion to new species have occurred in both older and newer models. The roles these new advances in modeling may have in future PD research are examined in this chapter.

Chapter 2 - Neurotoxic in vivo models of Parkinson’s disease: recent advances

Animal models have been invaluable to Parkinsons disease (PD) research. Of these, neurotoxin models have historically been the most widely utilized. The goal of this chapter is to give a brief historical description of classic PD models and then to identify the most recent important advances in modeling human PD in animals. Indeed, significant advances in modeling additional features of PD and expansion to new species have occurred in both older and newer models. The roles these new advances in modeling may have in future PD research are examined in this chapter.

Melatonin treatment potentiates neurodegeneration in a rat rotenone Parkinson's disease model

Parkinsons disease (PD) is characterized pathologically by progressive neurodegeneration of the nigrostriatal dopamine (DA) system. Currently, the cause of the disease is unknown, except for a small percentage of familial cases (<10% of total). The rat rotenone model reproduces many of the pathological features of the human disease, including apomorphine‐responsive behavioral deficits, DA depletion, loss of striatal DA terminals and nigral dopaminergic neurons, and α‐synuclein/polyubiquitin‐positive cytoplasmic inclusions reminiscent of Lewy bodies. Therefore, this model is well‐suited to examine potential neuroprotective agents. Melatonin is produced mainly by the pineal gland and is known primarily for regulating circadian rhythms. It also has potent free radical scavenging and antiinflammatory properties. Melatonin has been reported to be neuroprotective in the 6‐hydroxydopamine (6‐OHDA) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) models of PD. However, there are conflicting reports suggesting that melatonin does not provide neuroprotection in these models. Melatonin elicits significant functional changes in the nigrostriatal DA system that may affect 6‐OHDA and MPTP entry into cells. Therefore, rotenone is an ideal model for assessing protection, because it does not rely on the dopamine transporter uptake to exert neurotoxicity. In this study, the neuroprotective potential of melatonin in the rotenone PD model was assessed. Melatonin potentiated striatal catecholamine depletion, striatal terminal loss, and nigral DA cell loss. Indeed, melatonin alone elicited alterations in striatal catecholamine content. Our findings indicate that melatonin is not neuroprotective in the rotenone model of PD and may exacerbate neurodegeneration.

Lessons from the rotenone model of Parkinson's disease

In their recent review on environmental toxins and Parkinson’s disease (PD)1, under the heading ‘What have we learned from the Rotenone model of PD?’, Cicchetti and colleagues do not list a single positive aspect of the model. It is possible, however, to view the rotenone model from a different perspective. Rotenone was first used to model PD in 1985 when Heikkila injected this mitochondrial complex I inhibitor directly into the brain and showed that at a concentration of 5 mM – approximately 500,000-fold higher than its IC50 of 10 nM – it killed dopaminergic neurons2; however, identical results could have been obtained with a high concentration of virtually any toxin, mitochondrial or otherwise. Later, because there was a growing suspicion that PD might be associated with systemic mitochondrial defects, several groups began to experiment with systemic administration of mitochondrial toxins. Ferrante (1997) reported that administration of rotenone (10-18 mg/kg/day) produced ‘nonspecific’ brain lesions and peripheral toxicity3. In contrast, when Betarbet (2000) titrated the experimental complex I inhibition to a level similar to that reported in platelets from PD patients (using 2-3 mg/kg/day), it produced highly selective nigrostriatal degeneration4. Even more remarkably, rotenone-treated rats developed alpha-synuclein-positive cytoplasmic inclusions, similar to Lewy bodies, in nigral dopaminergic neurons – overcoming for the first time a key limitation of other available in vivo models. Moreover, rotenone provided the first proof-of-concept that a systemic defect in mitochondrial function could lead to selective nigrostriatal neurodegeneration. And although the rotenone model was developed initially to test the ‘mitochondrial hypothesis’ of PD, given the epidemiological links to pesticide exposure, it was also of interest that rotenone is a pesticide. Subsequent studies found that the rotenone model accurately recapitulates many other features of PD5, including: accumulation and aggregation of endogenous, wildtype alpha-synuclein; α-synuclein- and polyubiquitin-positive Lewy bodies and Lewy neurites; apomorphine-responsive behavioral deficits; early and sustained activation of microglia; oxidative modification and translocation of DJ-1 into mitochondria in vivo; impairment of the nigral ubiquitin-proteasome system; accumulation of iron in the substantia nigra through a mechanism involving transferrin and transferrin receptor 2; α-synuclein pathology in enteric neurons and functional deficits in GI function, including gastroparesis. Additionally, systemic rotenone treatment can recapitulate the retinal pathology, the loss of testosterone and some of the sleep disturbances that are typical of PD. It is also worth noting that the transferrin-dependent mechanism of iron accumulation discovered in the rotenone model was subsequently found to be operative in human PD6. In other words, the rotenone model predicted what would be found in PD. Further, after the initial work in rats, others reported successful application of the rotenone model in species that are more tractable genetically, including mice, Drosophila and C. elegans. It is also worth noting that, based on initial reports of rotenone neurotoxicity and the fact that it is a pesticide, epidemiological studies began to look at the potential role of exposure to rotenone per se as a risk factor for development of human PD. While the number of individuals exposed to rotenone and other ‘botanicals’ is small compared to other classes of synthetic pesticides, a recent study found an odds ratio of 5.9 (CI 0.6-56.1)7 and another found an odds ratio for rotenone of 10.9 (CI 2.5-48.0)8. Thus, the rotenone model informed subsequent epidemiological studies, which have suggested a potential role for rotenone in some cases of PD. For all its strengths, however, the rotenone model has limitations. As expected, this mitochondrial poison, like any other, can produce dose-dependent systemic toxicity and mortality. Moreover, especially when delivered by osmotic minipump, there has been substantial variability in the proportion of animals that become parkinsonian and in the extent of their nigrostriatal lesions. Despite this variability, many groups have reported that rotenone (2-3 mg/kg/day) produces selective nigrostriatal degeneration, generally without nonspecific lesions (e.g., see Fleming9). Nevertheless, for unclear reasons, a few labs have reported striatal or other lesions with systemic rotenone. For example, Cicchetti and colleagues (2004) found that rotenone caused “severe digestion problems” with a stomach that was enlarged and full of undigested food10. Although the authors may not have recognized it as such, this may have been the first indication that rotenone can reproduce gastrointestinal features of PD, such as gastroparesis. In fact, Drolet (2009) recently reported that rotenone accurately recapitulates pathological and functional features of parkinsonian gastrointestinal impairment11. Although the reasons for the discrepancies between labs are uncertain, recent refinements of the rotenone model have apparently made it more reproducible and have reduced nonspecific toxicities12. In the end, it is unrealistic to expect to be able to model perfectly in rats all aspects of an age-related disease like PD. Even genetically accurate models PD have met with limited success in replicating key behavioral and pathological features of the disease. Nevertheless, a great deal has been learned – and remains to be discovered – about pathogenic mechanisms using the rotenone model of PD.

Gene-environment interactions in Parkinson's disease: Specific evidence in humans and mammalian models

Interactions between genetic factors and environmental exposures are thought to be major contributors to the etiology of Parkinsons disease. While such interactions are poorly defined and incompletely understood, recent epidemiological studies have identified specific interactions of potential importance to human PD. In this review, the most current data on gene-environment interactions in PD from human studies are critically discussed. Animal models have also highlighted the importance of genetic susceptibility to toxicant exposure and data of potential relevance to human PD are discussed. Goals and needs for the future of the field are proposed.

shRNA targeting α-synuclein prevents neurodegeneration in a Parkinson's disease model.

Multiple convergent lines of evidence implicate both α-synuclein (encoded by SCNA) and mitochondrial dysfunction in the pathogenesis of sporadic Parkinsons disease (PD). Occupational exposure to the mitochondrial complex I inhibitor rotenone increases PD risk; rotenone-exposed rats show systemic mitochondrial defects but develop specific neuropathology, including α-synuclein aggregation and degeneration of substantia nigra dopaminergic neurons. Here, we inhibited expression of endogenous α-synuclein in the adult rat substantia nigra by adeno-associated virus-mediated delivery of a short hairpin RNA (shRNA) targeting the endogenous rat Snca transcript. Knockdown of α-synuclein by ~35% did not affect motor function or cause degeneration of nigral dopaminergic neurons in control rats. However, in rotenone-exposed rats, progressive motor deficits were substantially attenuated contralateral to α-synuclein knockdown. Correspondingly, rotenone-induced degeneration of nigral dopaminergic neurons, their dendrites, and their striatal terminals was decreased ipsilateral to α-synuclein knockdown. These data show that α-synuclein knockdown is neuroprotective in the rotenone model of PD and indicate that endogenous α-synuclein contributes to the specific vulnerability of dopaminergic neurons to systemic mitochondrial inhibition. Our findings are consistent with a model in which genetic variants influencing α-synuclein expression modulate cellular susceptibility to environmental exposures in PD patients. shRNA targeting the SNCA transcript should be further evaluated as a possible neuroprotective therapy in PD.

Pseudotype-dependent lentiviral transduction of astrocytes or neurons in the rat substantia nigra

Gene transfer to the central nervous system provides powerful methodology for the study of gene function and gene-environment interactions in vivo, in addition to a vehicle for the delivery of therapeutic transgenes for gene therapy. The aim of the present study was to determine patterns of tropism exhibited by pseudotyped lentiviral vectors in the rat substantia nigra, in order to evaluate their utility for gene transfer in experimental models of Parkinsons disease. Isogenic lentiviral vector particles encoding a GFP reporter were pseudotyped with envelope glycoproteins derived from vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), or Moloney murine leukemia virus (MuLV). Adult male Lewis rats received unilateral stereotactic infusions of vector into the substantia nigra; three weeks later, patterns of viral transduction were determined by immunohistological detection of GFP. Different pseudotypes gave rise to transgene expression in restricted and distinct cellular populations. VSV and MV pseudotypes transduced midbrain neurons, including a subset of nigral dopaminergic neurons. In contrast, LCMV- and MuLV-pseudotyped lentivirus produced transgene expression exclusively in astrocytes; the restricted transduction of astroglial cells was not explained by the cellular distribution of receptors previously shown to mediate entry of LCMV or MuLV. These data suggest that pseudotyped lentiviral vectors will be useful for experimental gene transfer to the rat substantia nigra. In particular, the availability of neuronal and astrocytic-targeting vectors will allow dissociation of cell autonomous and cell non-autonomous functions of key gene products in vivo.