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
Chronic consumption of β-sitosterol-β-D-glucoside (BSSG), a neurotoxin contained in cycad seeds, leads to Parkinson’s disease in humans and rodents. Here, we explored whether a single intranigral administration of BSSG triggers neuroinflammation and neurotoxic A1 reactive astrocytes besides dopaminergic neurodegeneration. We injected 6 μg BSSG/1 μL DMSO or vehicle into the left substantia nigra and immunostained with antibodies against tyrosine hydroxylase (TH) together with markers of microglia (OX42), astrocytes (GFAP, S100β, C3), and leukocytes (CD45). We also measured nitric oxide (NO), lipid peroxidation (LPX), and proinflammatory cytokines (TNF-α, IL-1β, IL-6). The Evans blue assay was used to explore the blood-brain barrier (BBB) permeability. We found that BSSG activates NO production on days 15 and 30 and LPX on day 120. Throughout the study, high levels of TNF-α were present in BSSG-treated animals, whereas IL-1β was induced until day 60 and IL-6 until day 30. Immunoreactivity of activated microglia (
899.0
±
80.20
%
) and reactive astrocytes (
651.50
±
11.28
%
) progressively increased until day 30 and then decreased to remain
251.2
±
48.8
%
(microglia) and
91.02
±
39.8
(astrocytes) higher over controls on day 120. C3(+) cells were also GFAP and S100β immunoreactive, showing they were neurotoxic A1 reactive astrocytes. BBB remained permeable until day 15 when immune cell infiltration was maximum. TH immunoreactivity progressively declined, reaching
83.6
±
1.8
%
reduction on day 120. Our data show that BSSG acute administration causes chronic neuroinflammation mediated by activated microglia, neurotoxic A1 reactive astrocytes, and infiltrated immune cells. The severe neuroinflammation might trigger Parkinson’s disease in BSSG intoxication.
Neurotensin (NTS)-polyplex is a multicomponent
nonviral vector
that enables gene delivery via internalization of the neurotensin
type 1 receptor (NTSR1) to dopaminergic neurons and cancer cells.
An approach to improving its therapeutic safety is replacing the viral
karyophilic component (peptide KPSV40; MAPTKRKGSCPGAAPNKPK), which
performs the nuclear import activity, by a shorter synthetic peptide
(KPRa; KMAPKKRK). We explored this issue and the mechanism of plasmid
DNA translocation through the expression of the green fluorescent
protein or red fluorescent protein fused with KPRa and internalization
assays and whole-cell patch-clamp configuration experiments in a single
cell together with importin α/β pathway blockers. We showed
that KPRa electrostatically bound to plasmid DNA increased the transgene
expression compared with KPSV40 and enabled nuclear translocation
of KPRa-fused red fluorescent proteins and plasmid DNA. Such translocation
was blocked with ivermectin or mifepristone, suggesting importin α/β
pathway mediation. KPRa also enabled NTS-polyplex-mediated expression
of reporter or physiological genes such as human mesencephalic-derived
neurotrophic factor (hMANF) in dopaminergic neurons in vivo. KPRa
is a synthetic monopartite peptide that showed nuclear import activity
in NTS-polyplex vector-mediated gene delivery. KPRa could also improve
the transfection of other nonviral vectors used in gene therapy.
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