Dose-related biphasic effect of the Parkinson's disease neurotoxin MPTP, on the spread, accumulation, and toxicity of α-synuclein.

Parkinson's disease (PD), the second most common progressive neurodegenerative disorder, is characterized by the abnormal accumulation of intraneuronal inclusions enriched in aggregated α-synuclein (α-syn), known as Lewy bodies (LBs) and Lewy neurites (LNs), and significant loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the brain. Recent evidence suggests that the intrastriatal inoculation of α-syn preformed fibrils (PFF) in mice brain triggers endogenous α-syn in interconnected brain regions. 1-methyl, 4-phenyl, 1,2,3,6 tetrahydropyridine (MPTP), a mitochondrial neurotoxin, has been used previously to generate a PD mouse model. However, the common methods of MPTP exposure do not induce LB or α-syn aggregation in mice. In the present study, we evaluated the effect of different doses of MPTP (10 mg/kg.b.wt and/or 25 mg/kg.b.wt) on the spread, accumulation, and toxicity of endogenous α-syn in mice administered an intrastriatal injection of human α-syn PFF. We inoculated human WT α-syn PFF in mouse striatum. At 6 weeks post PFF injection, we challenged the animal with two different doses of MPTP (10 mg/kg.b.wt and 25 mg/kg.b.wt) once daily for five consecutive days. At 2 weeks from the start of the MPTP regimen, we collected the mice brain and performed immunohistochemical analysis, and Rotarod test to assess motor coordination and muscle strength before and after MPTP injection. A single injection of human WT α-syn PFF in the mice striatum induced the propagation of α-syn, occurring as phosphorylated α-synuclein (pS129), towards the SNpc, within a very short time. Injection of a low dose of MPTP (10 mg/kg.b.wt) at 6 weeks post α-syn PFF inoculation further enhanced the spread, whereas a high dose of MPTP (25 mg/kg.b.wt.) reduced the spread. Majority of the accumulated α-syn were proteinase K resistant, as recognized using a conformation-specific α-syn antibody. Injection of α-syn PFF alone caused 12 % reduction in the number of tyrosine hydroxylase positive neurons while α-syn PFF + a low dose of MPTP caused 33 % reduction (loss), compared to the control mice injected with saline. This combination also reduced the motor coordination. Interestingly, a low dose of MPTP alone did not cause any significant reduction in the number of tyrosine hydroxylase positive neurons compared to saline treatment. Animals that received α-syn PFF and a high dose of MPTP showed massive activation of glial cells and decreased spread of α-syn, majority of which were detected in the nucleus. Our results suggest that a combination of human WT α-syn PFF and a low dose of MPTP increases the pathological conversion and propagation of endogenous α-syn, and neurodegeneration, within a very short time. Our model can be used to study the mechanisms of α-syn propagation and screen for potential drugs against PD.

Merghani MM ，Ardah MT ，Al Shamsi M ，Kitada T ，Haque ME ... -  《-》

Intrastriatal injection of pre-formed mouse α-synuclein fibrils into rats triggers α-synuclein pathology and bilateral nigrostriatal degeneration.

Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinson's disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague-Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180 days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting that PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time-point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180 days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180 days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.

Paumier KL ，Luk KC ，Manfredsson FP ，Kanaan NM ，Lipton JW ，Collier TJ ，Steece-Collier K ，Kemp CJ ，Celano S ，Schulz E ，Sandoval IM ，Fleming S ，Dirr E ，Polinski NK ，Trojanowski JQ ，Lee VM ，Sortwell CE ... -  《-》

Developmental exposure to the organochlorine pesticide dieldrin causes male-specific exacerbation of α-synuclein-preformed fibril-induced toxicity and motor deficits.

Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the α-synuclein (α-syn)-preformed fibril (PFF) model, which better reflects the α-syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to α-syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal α-syn PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated α-syn aggregation (pSyn) in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific exacerbation of synucleinopathy-induced behavioral and biochemical deficits. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD.

Gezer AO ，Kochmanski J ，VanOeveren SE ，Cole-Strauss A ，Kemp CJ ，Patterson JR ，Miller KM ，Kuhn NC ，Herman DE ，McIntire A ，Lipton JW ，Luk KC ，Fleming SM ，Sortwell CE ，Bernstein AI ... -  《-》

Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration.

Duffy MF ，Collier TJ ，Patterson JR ，Kemp CJ ，Luk KC ，Tansey MG ，Paumier KL ，Kanaan NM ，Fischer DL ，Polinski NK ，Barth OL ，Howe JW ，Vaikath NN ，Majbour NK ，El-Agnaf OMA ，Sortwell CE ... -  《Journal of Neuroinflammation》

PET imaging reveals early and progressive dopaminergic deficits after intra-striatal injection of preformed alpha-synuclein fibrils in rats.

Alpha-synuclein (a-syn) can aggregate and form toxic oligomers and insoluble fibrils which are the main component of Lewy bodies. Intra-neuronal Lewy bodies are a major pathological characteristic of Parkinson's disease (PD). These fibrillar structures can act as seeds and accelerate the aggregation of monomeric a-syn. Indeed, recent studies show that injection of preformed a-syn fibrils (PFF) into the rodent brain can induce aggregation of the endogenous monomeric a-syn resulting in neuronal dysfunction and eventual cell death. We injected 8 μg of murine a-syn PFF, or soluble monomeric a-syn into the right striatum of rats. The animals were monitored behaviourally using the cylinder test, which measures paw asymmetry, and the corridor task that measures lateralized sensorimotor response to sugar treats. In vivo PET imaging was performed after 6, 13 and 22 weeks using [11C]DTBZ, a marker of the vesicular monoamine 2 transporter (VMAT2), and after 15 and 22 weeks using [11C]UCB-J, a marker of synaptic SV2A protein in nerve terminals. Histology was performed at the three time points using antibodies against dopaminergic markers, aggregated a-syn, and MHCII to evaluate the immune response. While the a-syn PFF injection caused only mild behavioural changes, [11C]DTBZ PET showed a significant and progressive decrease of VMAT2 binding in the ipsilateral striatum. This was accompanied by a small progressive decrease in [11C]UCB-J binding in the same area. In addition, our histological analysis revealed a gradual spread of misfolded a-syn pathology in areas anatomically connected to striatum that became bilateral with time. The striatal a-syn PFF injection resulted in a progressive unilateral degeneration of dopamine terminals, and an early and sustained presence of MHCII positive ramified microglia in the ipsilateral striatum and substantia nigra. Our study shows that striatal injections of a-syn fibrils induce progressive pathological synaptic dysfunction prior to cell death that can be detected in vivo with PET. We confirm that intrastriatal injection of a-syn PFFs provides a model of progressive a-syn pathology with loss of dopaminergic and synaptic function accompanied by neuroinflammation, as found in human PD.

Thomsen MB ，Ferreira SA ，Schacht AC ，Jacobsen J ，Simonsen M ，Betzer C ，Jensen PH ，Brooks DJ ，Landau AM ，Romero-Ramos M ... -  《-》