Sheila M. Fleming

Parkin-deficient Mice Exhibit Nigrostriatal Deficits but Not Loss of Dopaminergic Neurons

Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinsons disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinsons disease, we generated a mouse model bearing a germline disruption in parkin. Parkin–/– mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin–/– mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin–/– mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin–/– mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinsons disease. Steady-state levels of CDCrel-1, synphilin-1, and α-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin–/– brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice.

A Neurovascular Niche for Neurogenesis after Stroke

Stroke causes cell death but also birth and migration of new neurons within sites of ischemic damage. The cellular environment that induces neuronal regeneration and migration after stroke has not been defined. We have used a model of long-distance migration of newly born neurons from the subventricular zone to cortex after stroke to define the cellular cues that induce neuronal regeneration after CNS injury. Mitotic, genetic, and viral labeling and chemokine/growth factor gain- and loss-of-function studies show that stroke induces neurogenesis from a GFAP-expressing progenitor cell in the subventricular zone and migration of newly born neurons into a unique neurovascular niche in peri-infarct cortex. Within this neurovascular niche, newly born, immature neurons closely associate with the remodeling vasculature. Neurogenesis and angiogenesis are causally linked through vascular production of stromal-derived factor 1 (SDF1) and angiopoietin 1 (Ang1). Furthermore, SDF1 and Ang1 promote post-stroke neuroblast migration and behavioral recovery. These experiments define a novel brain environment for neuronal regeneration after stroke and identify molecular mechanisms that are shared between angiogenesis and neurogenesis during functional recovery from brain injury.

Early and Progressive Sensorimotor Anomalies in Mice Overexpressing Wild-Type Human α-Synuclein

Accumulation of α-synuclein in brain is a hallmark of synucleinopathies, neurodegenerative diseases that include Parkinsons disease. Mice overexpressing α-synuclein under the Thy-1 promoter (ASO) show abnormal accumulation of α-synuclein in cortical and subcortical regions of the brain, including the substantia nigra. We examined the motor deficits in ASO mice with a battery of sensorimotor tests that are sensitive to alterations in the nigrostriatal dopaminergic system. Male wild-type and ASO mice were tested every 2 months for 8 months for motor performance and coordination on a challenging beam, inverted grid, and pole, sensorimotor deficits in an adhesive removal test, spontaneous activity in a cylinder, and gait. Fine motor skills were assessed by the ability to grasp cotton from a bin. ASO mice displayed significant impairments in motor performance and coordination and a reduction in spontaneous activity as early as 2 months of age. Motor performance and coordination impairments became progressively worse with age and sensorimotor deficits appeared at 6 months. Fine motor skills were altered at 4 months and worsened at 8 months. These data indicate that overexpression of α-synuclein induced an early and progressive behavioral phenotype that can be detected in multiple tests of sensorimotor function. These behavioral deficits provide a useful way to assess novel drug therapy in genetic models of synucleinopathies.

3,4-Dihydroxyphenylalanine Reverses the Motor Deficits in Pitx3-Deficient Aphakia Mice: Behavioral Characterization of a Novel Genetic Model of Parkinson's Disease

Parkinsons disease (PD) is a neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra. There is a need for genetic animal models of PD for screening and in vivo testing of novel restorative therapeutic agents. Although current genetic models of PD produce behavioral impairment and nigrostriatal dysfunction, they do not reproduce the loss of midbrain dopaminergic neurons and 3,4-dihydroxyphenylalanine (l-DOPA) reversible behavioral deficits. Here, we demonstrate that Pitx3-deficient aphakia (ak) mice, which have been shown previously to exhibit a major loss of substantia nigra dopaminergic neurons, display motor deficits that are reversed by l-DOPA and evidence of “dopaminergic supersensitivity” in the striatum. Thus, ak mice represent a novel genetic model exhibiting useful characteristics to test the efficacy of symptomatic therapies for PD and to study the functional changes in the striatum after dopamine depletion and l-DOPA treatment.

Genetic mouse models of Huntington's and Parkinson's diseases: illuminating but imperfect

Genetic mouse models based on identification of genes that cause Huntingtons and Parkinsons diseases have revolutionized understanding of the mechanistic pathophysiological progression of these disorders. These models allow the earliest manifestations of the diseases to be identified, and they display behavioral, neuropathological and electrophysiological deficits that can be followed over time in mechanistic and drug studies. An intriguing feature is that they do not reproduce the relatively selective and massive cell loss characterizing the human diseases. There is more information on Huntingtons disease models because the disorder involves a single gene that was identified over ten years ago; genetic mutations causing Parkinsons disease are rare and were discovered more recently, and models of the disease have been generated only within the past few years.

Genetic Mouse Models of Parkinsonism: Strengths and Limitations

SummaryParkinson’s disease (PD) is a progressive neurodegenerative disorder. Patients with PD display a combination of motor symptoms including resting tremor, rigidity, bradykinesia, and postural instability that worsen over time. These motor symptoms are related to the progressive loss of dopamine neurons in the substantia nigra pars compacta. PD patients also suffer from nonmotor symptoms that may precede the cardinal motor symptoms and that are likely related to pathology in other brain regions. Traditional toxin models of PD have focused on the nigrostriatal pathway and the loss of dopamine neurons in this region, and these models have been important in our understanding of PD and in the development of symptomatic treatments for the disease. However, they are limited in that they do not reproduce the full pathology and progression seen in PD, thus creating a need for better models. The recent discovery of specific genes causing familial forms of PD has contributed to the development of novel genetic mouse models of PD. This review discusses the validity, benefits, and limitations of these new models.

Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone

Mitochondrial toxins such as the complex 1 inhibitor rotenone are widely used as pesticides and may be present in military environments. Administration of rotenone can induce biochemical and histological alterations similar to those of Parkinsons disease in rats. However, only a subset of animals show these effects and it is unclear whether more subtle alterations are caused by chronic administration of rotenone in those animals that appear resistant to its toxic effects on dopaminergic nerve terminals. To address this question, vehicle or rotenone (2.0, 2.5, or 3.5 mg/kg/day) was administered intravenously or subcutaneously for 21 days to adult rats, and rotenone effects on survival, motor behavior, and striatal tyrosine hydroxylase immunoreactivity (TH-IR) were examined. Both intravenous and subcutaneous rotenone induced a dose-dependent decrease in survival rates. Surviving animals showed a decrease in spontaneous rearing. Locomotor activity and movement initiation time were also altered in some of the experimental groups. Confirming previous results, TH-IR in the striatum was markedly decreased in rats that fell ill early in the study and in a few of the surviving rats with high rotenone doses. However, none of the surviving rats receiving 2.0 mg/kg/day showed TH-IR loss reminiscent of Parkinsons disease, and loss of striatal TH-IR across doses was not correlated with motor behavior in individual rats. Thus, chronic administration of low doses of rotenone induces motor anomalies even in animals that do not develop histological signs of Parkinsons disease, indicating a pervasive neurological effect of moderate mitochondrial dysfunction in vivo.

Olfactory deficits in mice overexpressing human wildtype α-synuclein

Accumulation of α‐synuclein in neurons of the central and peripheral nervous system is a hallmark of sporadic Parkinson’s disease (PD) and mutations that increase α‐synuclein levels cause familial PD. Transgenic mice overexpressing α‐synuclein under the Thy1 promoter (Thy1‐aSyn) have high levels of α‐synuclein expression throughout the brain but no loss of nigrostriatal dopamine neurons up to 8 months, suggesting that they may be useful to model pre‐clinical stages of PD. Olfactory dysfunction often precedes the onset of the cardinal motor symptoms of PD by several years and includes deficits in odor detection, discrimination and identification. In the present study, we measured olfactory function in 3‐ and 9‐month‐old male Thy1‐aSyn mice with a buried pellet test based on latency to find an exposed or hidden odorant, a block test based on exposure to self and non‐self odors, and a habituation/dishabituation test based on exposure to non‐social odors. In a separate group of mice, α‐synuclein immunoreactivity was assessed in the olfactory bulb. Compared with wildtype littermates, Thy1‐aSyn mice could still detect and habituate to odors but showed olfactory impairments in aspects of all three testing paradigms. Thy1‐aSyn mice also displayed proteinase K‐resistant α‐synuclein inclusions throughout the olfactory bulb. These data indicate that overexpression of α‐synuclein is sufficient to cause olfactory deficits in mice similar to that observed in patients with PD. Furthermore, the buried pellet and block tests provided sufficient power for the detection of a 50% drug effect, indicating their usefulness for testing novel neuroprotective therapies.

HCN channelopathy in external globus pallidus neurons in models of Parkinson's disease

Parkinsons disease is a common neurodegenerative disorder characterized by a profound motor disability that is traceable to the emergence of synchronous, rhythmic spiking in neurons of the external segment of the globus pallidus (GPe). The origins of this pathophysiology are poorly defined for the generation of pacemaking. After the induction of a parkinsonian state in mice, there was a progressive decline in autonomous GPe pacemaking, which normally serves to desynchronize activity. The loss was attributable to the downregulation of an ion channel that is essential in pacemaking, the hyperpolarization and cyclic nucleotide–gated (HCN) channel. Viral delivery of HCN2 subunits restored pacemaking and reduced burst spiking in GPe neurons. However, the motor disability induced by dopamine (DA) depletion was not reversed, suggesting that the loss of pacemaking was a consequence, rather than a cause, of key network pathophysiology, a conclusion that is consistent with the ability of L-type channel antagonists to attenuate silencing after DA depletion.

Bacterial Artificial Chromosome Transgenic Mice Expressing a Truncated Mutant Parkin Exhibit Age-Dependent Hypokinetic Motor Deficits, Dopaminergic Neuron Degeneration, and Accumulation of Proteinase K-Resistant α-Synuclein

Recessive mutations in parkin are the most common cause of familial early-onset Parkinsons disease (PD). Recent studies suggest that certain parkin mutants may exert dominant toxic effects to cultured cells and such dominant toxicity can lead to progressive dopaminergic (DA) neuron degeneration in Drosophila. To explore whether mutant parkin could exert similar pathogenic effects to mammalian DA neurons in vivo, we developed a BAC (bacterial artificial chromosome) transgenic mouse model expressing a C-terminal truncated human mutant parkin (Parkin-Q311X) in DA neurons driven by a dopamine transporter promoter. Parkin-Q311X mice exhibit multiple late-onset and progressive hypokinetic motor deficits. Stereological analyses reveal that the mutant mice develop age-dependent DA neuron degeneration in substantia nigra accompanied by a significant loss of DA neuron terminals in the striatum. Neurochemical analyses reveal a significant reduction of the striatal dopamine level in mutant mice, which is significantly correlated with their hypokinetic motor deficits. Finally, mutant Parkin-Q311X mice, but not wild-type controls, exhibit age-dependent accumulation of proteinase K-resistant endogenous α-synuclein in substantia nigra and colocalized with 3-nitrotyrosine, a marker for oxidative protein damage. Hence, our study provides the first mammalian genetic evidence that dominant toxicity of a parkin mutant is sufficient to elicit age-dependent hypokinetic motor deficits and DA neuron loss in vivo, and uncovers a causal relationship between dominant parkin toxicity and progressive α-synuclein accumulation in DA neurons. Our study underscores the need to further explore the putative link between parkin dominant toxicity and PD.