Unilateral intranigral administration of β-sitosterol β-D-glucoside triggers pathological α-synuclein spreading and bilateral nigrostriatal dopaminergic neurodegeneration in the rat

Abstract

The spreading and accumulation of α-synuclein and dopaminergic neurodegeneration, two hallmarks of Parkinson's disease (PD), have been faithfully reproduced in rodent brains by chronic, oral administration of β-sitosterol β-Dglucoside (BSSG). We investigated whether a single injection of BSSG (6 μg BSSG/μL DMSO) in the left substantia nigra of Wistar rats causes the same effects. Mock DMSO injections and untreated rats formed control groups. We performed immunostainings against the pathological α-synuclein, the dopaminergic marker tyrosine hydroxylase (TH), the neuroskeleton marker β-III tubulin, the neurotensin receptor type 1 (NTSR1) as non-dopaminergic phenotype marker and Fluro-Jade C (F-J C) label for neurodegeneration. Using β-galactosidase (β-Gal) assay and active caspase-3 immunostaining, we assessed cell death mechanisms. Golgi-Cox staining was used to measure the density and types of dendritic spines of striatal medium spiny neurons. Motor and non-motor alterations were also evaluated. The study period comprised 15 to 120 days after the lesion. In the injured substantia nigra, BSSG caused a progressive α-synuclein aggregation and dopaminergic neurodegeneration caused by senescence and apoptosis. The α-synuclein immunoreactivity was also present within microglia cells. Decreased density of dopaminergic fibers

Trans-synaptic and retrograde axonal spread of Lewy pathology following pre-formed fibril injection in an in vivo A53T alpha-synuclein mouse model of synucleinopathy

“…One particular question of interest in relation to the trans-synaptic spread and retrograde propagation of Lewy pathology that we detect in our A53T SynGFP model after alpha-synuclein PFF injection is whether other, non-PFF-based models of progressive alpha-synuclein aggregation exhibit similar mechanisms. Substantial work has been done characterizing progressive, potentially trans-synaptic, spread of alpha-synuclein aggregation after oral exposure of rodents to the neurotoxin BSSG [ 32 , 33 , 73 ] and formation of aggregates in dopamine neurons after administration of bacterial LPS with LPS-simulated autologous macrophages [ 67 ]. It will be important in future studies to determine whether these relevant, toxin-induced forms of parkinsonism use similar trans-synaptic and retrograde Lewy pathology propagation mechanisms.…”

Intranigral Administration of β-Sitosterol-β-D-Glucoside Elicits Neurotoxic A1 Astrocyte Reactivity and Chronic Neuroinflammation in the Rat Substantia Nigra

“…This pathological process seems to mediate the BSSG-triggered dopaminergic loss [32] because these neurons express NMDA receptors [54,55] and are sensitive to glutamate [56]. The increase in glutamate can be caused by pathological α-synuclein aggregates [57] known to occur in the acute [38] and chronic [35] BSSG administration. The sustained NO production that coincided with the periods of microglia activation, reactive astrocyte induction, and leukocyte infiltration can be due to the iNOS expression in those inflammatory cells [58,59].…”

“…However, this issue was not explored in SNpc injected because the profound dopaminergic neurodegeneration suggests that the contribution of anti-inflammatory cytokines might be smaller than that of proinflammatory cytokines. Similar to A2 reactive astrocytes, it would be relevant to study the balance between pro-and antiinflammatory cytokinesis to gain insight into the control of the immune response in those brain regions where neuroinflammation is emerging by the presence of pathological αsynuclein [37,38].…”

“…Endogenous peroxidase was eliminated by incubating the slices with 3% hydrogen peroxide in PBS/Triton and 10% methanol at RT for 10 min. The immunohistochemical staining was developed using the ABC kit (1,10; Vector Laboratories; Burlingame, CA, USA) and 3′3-diaminobenzidine (DAB; Sigma-Aldrich; St. Louis, MO, USA) as reported previously [38]. The brain slices were washed 3 times for 5 min in PBS, counterstained with β-Gal and mounted on slides using Entellan resin (Merck, KGaA; Darmstadt, Germany), and observed with a light Leica DMIRE2 microscope with 63x (oil immersion) objective (Leica Microsystems; Nussloch, Germany).…”

“…However, whether BSSG administration could lead to reactive astrocyte induction, leukocyte infiltration, and production of chemical mediators of inflammation is still unknown. We recently showed that a single intranigral administration of BSSG reproduces, in less time, most of the features of oral administration, including dopaminergic neuron loss and pathological α-synuclein aggregation in the SNpc [37,38].…”