Although α-synuclein (α-SYN) aggregation is a hallmark of sporadic and familial Parkinson's disease (PD), it is not known how it contributes to early events of PD pathogenesis such as oxidative and inflammatory stress. Here, we addressed this question in a new animal model based on stereotaxic delivery of an adeno-associated viral vector (rAAV) for expression of human α-SYN in the ventral midbrain of mice lacking the transcription factor Nrf2 (Nrf2^−/−). Two months after surgery, Nrf2^−/− mice exhibited exacerbated degeneration of nigral dopaminergic neurons and increased dystrophic dendrites, reminiscent of Lewy neurites, which correlated with impaired proteasome gene expression and activity. Dopaminergic neuron loss was associated with an increase in neuroinflammation and gliosis that were intensified in Nrf2^−/− mice. In response to exogenously added α-SYN, Nrf2^−/− microglia failed to activate the expression of two anti-inflammatory genes, heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate quinone oxidorreductase-1 (NQO1). This impaired Nrf2 response correlated with a shift in the microglial activation profile, towards increased production of proinflammatory markers, IL-6, IL-1β and iNOS and reduced phagocytic capacity of fluorescent beads, and lower messenger RNA levels for TAM receptors Axl and Mer. Postmortem brain tissue samples from patients in early- to middle-stage progression of PD showed increased HO-1 expression in astrocytes and microglia, suggesting an attempt of the diseased brain to compensate these hallmarks of PD through activation of the Nrf2 pathway. This study demonstrates that α-SYN and Nrf2 deficiency cooperate on protein aggregation, neuroinflammation and neuronal death and provides a bifactorial animal model to study early-stage PD.

Parkinson’s disease (PD) is a complex, chronic, and progressive neurodegenerative disease that is characterized by irreversible dopaminergic neuronal loss in the substantia nigra. Alpha-synuclein is normally a synaptic protein that plays a key role in PD due to pathological accumulation as oligomers or fibrils. Clustered alpha-synuclein binds to the Toll-like receptors and activates the microglia, which initiates a process that continues with pro-inflammatory cytokine production and secretion. Pro-inflammatory cytokine overproduction and secretion induce cell death and accelerate PD progression. Microglia are found in a resting state in physiological conditions. Microglia became activated by stimulating Toll-like receptors on it under pathological conditions, such as alpha-synuclein aggregation, environmental toxins, or oxidative stress. The interaction between Toll-like receptors and its downstream pathway triggers an activation series, leads to nuclear factor-kappa B activation, initiates the inflammasome formation, and increases cytokine levels. This consecutive inflammatory process leads to dopaminergic cell damage and cell death. Microglia become overactive in response to chronic inflammation, which is observed in PD and causes excessive cytotoxic factor production, such as reactive oxidase, nitric oxide, and tumor necrosis factor-alpha. This inflammatory process contributes to the exacerbation of pathology by triggering neuronal damage or death. Current treatments, such as dopaminergic agonists, anticholinergics, or monoamine oxidase inhibitors alleviate PD symptoms, but they can not stop the disease progression. Finding a radical treatment option or stopping the progression is essential when considering that PD is the second most reported neurodegenerative disorder. Many cytokines are released during inflammation, and they can start the phagocytic process, which caused the degradation of infected cells along with healthy ones. Therefore, targeting the pathological mechanisms, such as microglial activation, mitochondrial dysfunction, and oxidative stress, that should be involved in the treatment program is important. Neuroinflammation is one of the key factors involved in PD pathogenesis as well as alpha-synuclein accumulation, synaptic dysfunction, or dopaminergic neuronal loss, especially in the substantia nigra. Therefore, evaluating the therapeutic efficiency of the mechanisms is important, such as microglial activation and nuclear factor-kappa B pathway or inflammasome formation inhibition, and cytokine release interruption against neuroinflammation may create new treatment possibilities for PD. This study examined the pathological relation between PD and neuroinflammation, and targeting neuroinflammation as an opportunity for PD treatments, such as Toll-like receptor antagonists, NOD-like receptor family pyrin domain containing-3 inflammasome inhibitors, cytokine inhibitors, peroxisome proliferator-activated receptor-γ agonists, reactive oxygen species inhibitors, and nonsteroidal anti-inflammatory drugs.

Short-hairpin RNA (shRNA)–mediated gene knockdown is a powerful tool for targeted gene silencing and an emerging novel therapeutic strategy. Recent publications, however, reported unexpected toxicity after utilizing viral-mediated shRNA knockdown in vivo. Thus, it is currently unclear whether shRNA-mediated knockdown strategy can be used as a safe and efficient tool for gene silencing. In this study, we have generated rAAV vectors expressing shRNAs targeting the rat tyrosine hydroxylase (TH) mRNA (shTH) for testing the efficacy of in vivo TH knockdown in the nigral dopaminergic neurons. At high titers, not only the shTH vectors but also the scrambled and green fluorescence protein (GFP)–only controls caused cell death. In a dose–response study, we identified a dose window leading to >60% decrease in TH⁺ neurons without any change in vesicular monoamine transporter-2 (VMAT2) expression. Moreover, using the safe and efficient dose, we showed that dopamine (DA) synthesis rate was significantly reduced and this lead to emergence of motor deficits in the shTH-expressing rats. Interestingly, these animals showed very robust and long-lasting recovery after a single systemic L-3,4-dihydroxyphenylalanine (L-DOPA) administration beyond what can be achieved in 6-hydroxydopamine (6-OHDA)–lesioned rats. Our results have implications for both mechanistic and therapeutic studies utilizing long-term shRNA-mediated gene silencing in the nigrostriatal projection system.

Although Parkinson's disease is a movement disorder, in many patients cognitive dysfunction is an important clinical sign. It is not yet clear whether this is attributable solely to a decrease in dopamine levels, or whether other neurotransmitter systems might be involved as well. In the present study, the importance of the mesocorticolimbic dopamine pathway and a possible convergence with forebrain cholinergic projections to neocortex and hippocampus in the regulation of learning and memory abilities were investigated by using specific lesion paradigms in one or both systems. Lesioning of dopaminergic neurons in the ventral tegmental area resulted in an impaired performance in the reference memory task, whereas the execution of the working memory tasks appeared to be unaffected in the Morris water maze. Analysis of the swim paths revealed that the dopamine-depleted animals were capable of adapting a search strategy on a given testing day but failed to transfer this information to the next day, suggesting a deficit in information storage and/or recall. In contrast, cholinergic lesions alone were without effect in all test paradigms. However, when both dopamine and acetylcholine were depleted, animals were also impaired in the working memory task, indicating that a functional convergence of the inputs from these systems was critical for acquisition of spatial memory. Interestingly, such an additional acquisition deficit appeared only after hippocampal cholinergic depletion regardless of a concurrent disruption of basalo cortical cholinergic afferents. Thus, further analyses of cholinergic alterations may prove useful in better understanding the cognitive symptoms in Parkinson's disease.

Drug-induced dyskinesias in dopamine-denervated animals are known to depend on both pre- and postsynaptic changes of the nigrostriatal circuitry. In lesion models used thus far, changes occur in both of these compartments and, therefore, it has not been possible to dissect the individual contribution of each compartment in the pathophysiology of dyskinesias. Here we silenced the nigrostriatal dopamine neurotransmission without affecting the anatomical integrity of the presynaptic terminals using a short-hairpin RNA-mediated knockdown of tyrosine hydroxylase enzyme (shTH). This treatment resulted in significant reduction (by about 70%) in extracellular dopamine concentration in the striatum as measured by on-line microdialysis. Under these conditions, the animals remained nondyskinetic after chronic L-DOPA treatment, whereas partial intrastriatal 6-hydoxydopamine lesioned rats with comparable reduction in extracellular dopamine levels developed dyskinesias. On the other hand, apomorphine caused moderate to severe dyskinesias in both groups. Importantly, single-dose L-DOPA challenge in apomorphine-primed shTH animals failed to activate the already established abnormal postsynaptic responses. Taken together, these data provide direct evidence that the status of the presynaptic, DA releasing compartment is a critical determinant of both the induction and maintenance of L-DOPA–induced dyskinesias.