Impact of N-Acetyl-L-Cysteine on the Pathology of Experimental Parkinson’s Disease in Vivo

Parkinson’s disease is a progressive neurodegenerative disorder associated with disruptions in motor as well as non-motor functions, such as cognitive and olfactory impairments. Postmortem tissue from Parkinson’s patients shows evidence of oxidative stress in dopaminergic neurons and hallmark proteinaceous inclusions known as Lewy bodies in multiple brain regions spanning the medulla oblongata to the telencephalon. There are no therapies that decelerate the progression of this disease. Thus, the major goal of the present study was to test the therapeutic potential of two neuroprotective molecules, the antioxidant thiol N-acetyl-L-cysteine (NAC) and the steroid neuromodulator dehyroepiandrosterone sulfate (DHEAS), in experimental models of Parkinson’s disease in vivo. To accomplish this goal, we first established multiple animal models of Parkinson’s disease that mimicked oxidative and/or proteotoxic stress: the 6-hydroxydopamine (6-OHDA) model of dopaminergic neurodegeneration and the alpha-synuclein model of Lewy-like pathology. NAC offered only transient protection in the 6-OHDA model, as demonstrated by multiple histological techniques that were validated in the present study. Indeed, NAC was mildly toxic at doses previously employed in the clinic, with implications for the long-term use of NAC in patients with chronic neurodegenerative conditions. We also developed a model of Lewy-like pathology in the hippocampus in which to examine the effects of DHEAS upon memory function. Although DHEAS failed to affect memory, we subsequently discovered that infusions of waterbath-sonicated alpha-synuclein fibrils into hippocampal CA2/CA3 led to robust Lewy-like pathology in some (but not all) of the brain regions that send first-order efferent projections to the hippocampus—the amygdala, entorhinal cortex, and contralateral CA3. Similar to the human condition, we collected evidence of selective vulnerability to alpha-synucleinopathy, as the septohippocampal projections were spared in our model. Notably, Lewy-like pathology in the hippocampus was statistically correlated with memory and olfactory deficits. Taken together, these studies reveal a novel model of proteinopathy in the hippocampus, which is known to develop Lewy pathology at mid-to-end stages of Parkinson’s disease and may be partly responsible for cognitive deficits in this condition. This model can now be used to test neuroprotective drug candidates that have the potential to ameliorate proteinopathic stress and improve neurological outcomes

Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe

Supplemental files to the publication Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe : Supplemental information (PDF): Materials and methods, tables, and supplemental figures S1-S8 (all supplemental figures are mentioned in the main text). Two mp4 movie files showing perinuclear localization of pSer129 signal (red) around NeuN+ nuclei (green). One movie shows a rotating cell and in the other video, the red pSer129 signal is peeled away to reveal the underlying green NeuN+ nucleus. Four high resolution figures (TIFF files)

Investigation of the therapeutic potential of N-acetyl cysteine and the tools used to define nigrostriatal degeneration in vivo

The glutathione precursor N-acetyl-l-cysteine (NAC) is currently being tested on Parkinson\u27s patients for its neuroprotective properties. Our studies have shown that NAC can elicit protection in glutathione-independent manners in vitro. Thus, the goal of the present study was to establish an animal model of NAC-mediated protection in which to dissect the underlying mechanism. Mice were infused intrastriatally with the oxidative neurotoxicant 6-hydroxydopamine (6-OHDA; 4 μg) and administered NAC intraperitoneally (100 mg/kg). NAC-treated animals exhibited higher levels of the dopaminergic terminal marker tyrosine hydroxylase (TH) in the striatum 10d after 6-OHDA. As TH expression is subject to stress-induced modulation, we infused the tracer FluoroGold into the striatum to retrogradely label nigrostriatal projection neurons. As expected, nigral FluoroGold staining and cell counts of FluoroGold+ profiles were both more sensitive measures of nigrostriatal degeneration than measurements relying on TH alone. However, NAC failed to protect dopaminergic neurons 3 weeks following 6-OHDA, an effect verified by four measures: striatal TH levels, nigral TH levels, nigral TH+ cell counts, and nigral FluoroGold levels. Some degree of mild toxicity of FluoroGold and NAC was evident, suggesting that caution must be exercised when relying on FluoroGold as a neuron-counting tool and when designing experiments with long-term delivery of NAC-such as clinical trials on patients with chronic disorders. Finally, the strengths and limitations of the tools used to define nigrostriatal degeneration are discussed

Critical appraisal of pathology transmission in the α-synuclein fibril model of Lewy body disorders [Supplemental material]

Lewy body disorders are characterized by the emergence of α-synucleinopathy in many parts of the central and peripheral nervous systems, including in the telencephalon. Dense α-synuclein+ pathology appears in regio inferior of the hippocampus in both Parkinson’\u27s disease and dementia with Lewy bodies and may disturb cognitive function. The preformed α-synuclein fibril model of Parkinson’\u27s disease is growing in use, given its potential for seeding the self-propagating spread of α-synucleinopathy throughout the mammalian brain. Although it is often assumed that the spread occurs through neuroanatomical connections, this is generally not examined vis-à-vis the uptake and transport of tract-tracers infused at precisely the same stereotaxic coordinates. As the neuronal connections of the hippocampus are historically well defined, we examined the first-order spread of α- synucleinopathy three months following fibril infusions centered in the mouse regio inferior (CA2 + CA3), and contrasted this to retrograde and anterograde transport of the established tract-tracers FluoroGold and biotinylated dextran amines (BDA). Massive hippocampal α-synucleinopathy was insufficient to elicit memory deficits or loss of cells and synaptic markers in this model of early disease processes. However, dense α-synuclein+ inclusions in the fascia dentata were negatively correlated with memory capacity. A modest compensatory increase in synaptophysin was evident in the stratum radiatum of cornu Ammonis in fibril-infused animals, and synaptophysin expression correlated inversely with memory function in fibril but not PBS-infused mice. No changes in synapsin I/II expression were observed. The spread of α-synucleinopathy was somewhat, but not entirely consistent with FluoroGold and BDA axonal transport, suggesting that variables other than innervation density also contribute to the materialization of α-synucleinopathy. For example, layer II entorhinal neurons of the perforant pathway exhibited somal α-synuclein+ inclusions as well as retrogradely labeled FluoroGold+ somata. However, some afferent brain regions displayed dense retrograde FluoroGold label and no α-synuclein+ inclusions (e.g. medial septum/diagonal band), supporting the selective vulnerability hypothesis. The pattern of inclusions on the contralateral side was consistent with specific spread through commissural connections (e.g. stratum pyramidale of CA3), but again, not all commissural projections exhibited α-synucleinopathy (e.g. hilar mossy cells). The topographical extent of inclusions is displayed here in high-resolution images that afford viewers a rich opportunity to dissect the potential spread of pathology through neural circuitry. Finally, the results of this expository study were leveraged to highlight the challenges and limitations of working with preformed α-synuclein fibrils