Synthetic Monopartite Peptide That Enables the Nuclear Import of Genes Delivered by the Neurotensin-Polyplex Vector.

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/ß 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/ß 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.

Cerebral dopamine neurotrophic factor transfection in dopamine neurons using neurotensin-polyplex nanoparticles reverses 6-hydroxydopamine-induced nigrostriatal neurodegeneration.

Overexpression of neurotrophic factors in nigral dopamine neurons is a promising approach to reverse neurodegeneration of the nigrostriatal dopamine system, a hallmark in Parkinson's disease. The human cerebral dopamine neurotrophic factor (hCDNF) has recently emerged as a strong candidate for Parkinson's disease therapy. This study shows that hCDNF expression in dopamine neurons using the neurotensin-polyplex nanoparticle system reverses 6-hydroxydopamine-induced morphological, biochemical, and behavioral alterations. Three independent electron microscopy techniques showed that the neurotensin-polyplex nanoparticles containing the hCDNF gene, ranging in size from 20 to 150 nm, enabled the expression of a secretable hCDNF in vitro. Their injection in the substantia nigra compacta on day 21 after the 6-hydroxydopamine lesion resulted in detectable hCDNF in dopamine neurons, whose levels remained constant throughout the study in the substantia nigra compacta and striatum. Compared with the lesioned group, tyrosine hydroxylase-positive (TH+) nigral cell population and TH+ fiber density rose in the substantia nigra compacta and striatum after hCDNF transfection. An increase in ßIII-tubulin and growth-associated protein 43 phospho-S41 (GAP43p) followed TH+ cell recovery, as well as dopamine and its catabolite levels. Partial reversal (80%) of drug-activated circling behavior and full recovery of spontaneous motor and non-motor behavior were achieved. Brain-derived neurotrophic factor recovery in dopamine neurons that also occurred suggests its participation in the neurotrophic effects. These findings support the potential of nanoparticle-mediated hCDNF gene delivery to develop a disease-modifying treatment against Parkinson's disease. The Institutional Animal Care and Use Committee of Centro de Investigación y de Estudios Avanzados approved our experimental procedures for animal use (authorization No. 162-15) on June 9, 2019.

A sequential methodology for integral evaluation of motor and non-motor behaviors in parkinsonian rodents.

An animal model, suitable for resembling Parkinson's disease (PD) progress, should show both, motor and non-motor alterations. However, these features have been scarcely evaluated or developed in parkinsonian models induced by neurotoxins. This protocol provides modifications to original methods, allowing six different motor and non-motor behavior tests, which adequately and timely emulate the main parkinsonian sensorimotor alterations in the rat or mouse: (1) bilateral sensorimotor alterations, examined by the vibrissae test; (2) balance and motor coordination, evaluated by the uncoordinated gait test; (3) locomotor asymmetry, analyzed by the cylinder test; (4) bradykinesia, as a locomotor alteration evidenced by the open field test; (5) depressive-like behavior, judged by the forced swimming test; and (6) hyposmia, assessed by the olfactory asymmetry test. Some advantages of using these behavioral tests over others include:•No sophisticated materials or equipment are required for their application and evaluation.•They are used in rodent models for parkinsonian research, but they can also be helpful for studying other movement disorders.•These tests can accurately discriminate the affected side from the healthy one, after unilateral injury of one hemisphere, resulting in sensorimotor, olfactory or locomotor asymmetry.

A single intranigral administration of ß-sitosterol ß-d-glucoside elicits bilateral sensorimotor and non-motor alterations in the rat.

Parkinson's disease (PD) is a progressive neuropathology characterized by motor and non-motor alterations. ß-sitosterol ß-d-glucoside (BSSG) is a neurotoxin whose prolonged oral administration in rats has been proposed as a new PD model. Herein, we demonstrate that a single, unilateral, and intranigral administration of BSSG also elicits bilateral sensorimotor alterations in the rat. Six behavioral tests evaluated the effect of different concentrations of BSSG (3, 6, 9, and 12 µg/µL DMSO) from 15 to 120 days after administration. The first behavioral alterations, which appeared on day 15, were unbalanced and uncoordinated gaits and a decrease in the sensorimotor cortex activity, as evidenced by the beam-walking and the vibrissae tests, respectively. After 30 days, the corridor test revealed hyposmia and a decreased locomotor activity in the open field. The last alteration was a depressive-like behavior, as shown by the forced swim test on days 60 and 120. According to the cylinder test, no locomotor asymmetry was observed over time with any BSSG concentrations tested. Also, a mesencephalic TH(+) cell loss (p < 0.05) was shown on day 30 when compared with the mock condition, and such a loss was even higher on day 120. At this time, the presence of pathological α-synuclein aggregates in the mesencephalon was documented. Our results show that the stereotaxic intranigral administration of BSSG reproduces some characteristics of oral administration, such as the progression of behavioral alterations, dopaminergic neurons loss, and the presence of Lewy body-like synuclein aggregations, in less time and resources.

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

Chronic consumption of ß-sitosterol-ß-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, S100ß, C3), and leukocytes (CD45). We also measured nitric oxide (NO), lipid peroxidation (LPX), and proinflammatory cytokines (TNF-α, IL-1ß, 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-1ß 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 S100ß 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.

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

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 ß-sitosterol ß-D-glucoside (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 ß-III tubulin, the neurotensin receptor type 1 (NTSR1) as non-dopaminergic phenotype marker and Fluro-Jade C (F-J C) label for neurodegeneration. Using ß-galactosidase (ß-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 and dendritic spines also occurred in the striatum. Remarkably, all the histopathological changes also appeared on the contralateral nigrostriatal system, and α-synuclein aggregates were present in other brain regions. Motor and non-motor behavioral alterations were progressive. Our data show that the stereotaxic BSSG administration reproduces PD α-synucleinopathy phenotype in the rat. This approach will aid in identifying the spread mechanism of α-synuclein pathology and validate anti-synucleinopathy therapies.

Acute Neuroinflammatory Response in the Substantia Nigra Pars Compacta of Rats after a Local Injection of Lipopolysaccharide.

Models of Parkinson's disease with neurotoxins have shown that microglial activation does not evoke a typical inflammatory response in the substantia nigra, questioning whether neuroinflammation leads to neurodegeneration. To address this issue, the archetypal inflammatory stimulus, lipopolysaccharide (LPS), was injected into the rat substantia nigra. LPS induced fever, sickness behavior, and microglial activation (OX42 immunoreactivity), followed by astrocyte activation and leukocyte infiltration (GFAP and CD45 immunoreactivities). During the acute phase of neuroinflammation, pro- and anti-inflammatory cytokines (TNF-α, IL-1ß, IL-6, IL-4, and IL-10) responded differentially at mRNA and protein level. Increased NO production and lipid peroxidation occurred at 168 h after LPS injection. At this time, evidence of neurodegeneration could be seen, entailing decreased tyrosine hydroxylase (TH) immunoreactivity, irregular body contour, and prolongation discontinuity of TH+ cells, as well as apparent phagocytosis of TH+ cells by OX42+ cells. Altogether, these results show that LPS evokes a typical inflammatory response in the substantia nigra that is followed by dopaminergic neurodegeneration.

Effect of chemical composition on the induction of DNA damage by urban airborne particulate matter.

Airborne particulate matter (PM) contains a large number of genotoxic substances capable of endangering human health. In the present study, we have investigated the ability of chemically characterized water-soluble and organic-soluble fractions of two particle sizes (PM2.5 and PM10) from different regions of Mexico City to induce DNA damage in a human lung epithelial cell line. We also evaluated associations between the physicochemical parameters of the PM and its genotoxicity. The airborne particulate samples were collected from four regions of the city; a HiVol air sampler was used to collect PM10 on glass fiber filters and a tapered element oscillating system coupled to an automatic cartridge collection unit was used to collect PM2.5 on teflon filters. PM mass was determined by gravimetric analysis of the filters. Filters containing PM2.5 and one section of each PM10 filter were agitated either with deionized water to extract water-soluble compound, or with dichloromethane to prepare organic-soluble compounds. The chemical composition of the extracts was determined by ion and gas chromatography and atomic adsorption spectroscopy. A549 human type II alveolar epithelial cells were exposed to different concentrations of the PM2.5 and PM10 extracts, and alkaline single cell gel electrophoresis or the Comet assay was performed to measure DNA damage and repair. These analyses indicated that soluble transition metals and the organic-soluble PM fractions are crucial factors in the DNA damage induced by PM. PM composition was more important than PM mass for producing genotoxicity. The results of this study showed that the constituents of the water-soluble PM extract are more likely to induce DNA damage than the organic compounds.