Modulation of Tyrosine Hydroxylase, Neuropeptide Y, Glutamate, and Substance P in Ganglia and Brain Areas Involved in Cardiovascular Control after Chronic Exposure to Nicotine

Considering that nicotine instantly interacts with central and peripheral nervous systems promoting cardiovascular effects after tobacco smoking, we evaluated the modulation of glutamate, tyrosine hydroxylase (TH), neuropeptide Y (NPY), and substance P (SP) in nodose/petrosal and superior cervical ganglia, as well as TH and NPY in nucleus tractus solitarii (NTS) and hypothalamic paraventricular nucleus (PVN) of normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after 8 weeks of nicotine exposure. Immunohistochemical and in situ hybridization data demonstrated increased expression of TH in brain and ganglia related to blood pressure control, preferentially in SHR, after nicotine exposure. The alkaloid also increased NPY immunoreactivity in ganglia, NTS, and PVN of SHR, in spite of decreasing its receptor (NPY1R) binding in NTS of both strains. Nicotine increased SP and glutamate in ganglia. In summary, nicotine positively modulated the studied variables in ganglia while its central effects were mainly constrained to SHR

Blast waves from detonated military explosive reduce GluR1 and synaptophysin levels in hippocampal slice cultures

Explosives create shockwaves that cause blast-induced neurotrauma, one of the most common types of traumatic brain injury (TBI) linked to military service. Blast-induced TBIs are often associated with reduced cognitive and behavioral functions due to a variety of factors. To study the direct effects of military explosive blasts on brain tissue, we removed systemic factors by utilizing rat hippocampal slice cultures. The long-term slice cultures were briefly sealed air-tight in serum-free medium, lowered into a 37 °C water-filled tank, and small 1.7-gram assemblies of cyclotrimethylene trinitramine (RDX) were detonated 15 cm outside the tank, creating a distinct shockwave recorded at the culture plate position. Compared to control mock-treated groups of slices that received equal submerge time, 1–3 blast impacts caused a dose-dependent reduction in the AMPA receptor subunit GluR1. While only a small reduction was found in hippocampal slices exposed to a single RDX blast and harvested 1–2 days later, slices that received two consecutive RDX blasts 4 min apart exhibited a 26–40% reduction in GluR1, and the receptor subunit was further reduced by 64–72% after three consecutive blasts. Such loss correlated with increased levels of HDAC2, a histone deacetylase implicated in stress-induced reduction of glutamatergic transmission. No evidence of synaptic marker recovery was found at 72 h post-blast. The presynaptic marker synaptophysin was found to have similar susceptibility as GluR1 to the multiple explosive detonations. In contrast to the synaptic protein reductions, actin levels were unchanged, spectrin breakdown was not detected, and Fluoro-Jade B staining found no indication of degenerating neurons in slices exposed to three RDX blasts, suggesting that small, sub-lethal explosives are capable of producing selective alterations to synaptic integrity. Together, these results indicate that blast waves from military explosive cause signs of synaptic compromise without producing severe neurodegeneration, perhaps explaining the cognitive and behavioral changes in those blast-induced TBI sufferers that have no detectable neuropathology

Aβ42-mediated proteasome inhibition and associated tau pathology in hippocampus are governed by a lysosomal response involving cathepsin B: Evidence for protective crosstalk between protein clearance pathways.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer's disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aβ42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aβ42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aβ42 (0.5-1.5 μM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aβ42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aβ42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aβ42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aβ42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aβ42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways

Pre-aggregated Aβ₄₂ causes proteasomal dysfunction in correspondence with synaptic decline in rat hippocampal slice cultures.

<p>The brain tissue was maintained on culture inserts with media placed below the insert membrane—note the two Nissl-stained slices showing their insert positions and neuronal layers (a) (size bar = 3 mm). After several weeks in culture, a hippocampal slice was stained with anti-synaptophysin and DAPI to show the stable maintenance of CA1, CA3, and dentate gyrus subfields and their associated dense neuropil (b) (view-field width: 2.6 mm). Aliquots of pre-aggregated, human Aβ₄₂ peptide were diluted to 0.5–1.5 μM and applied daily to slice cultures alongside vehicle-treated slices. Proteasome chymotrypsin-like activity (mean Vmax/s ± SEM normalized to control; n = 6) was measured in hippocampal slices that were harvested after 4–6 days of treatment (c). For immunoblots, groups of 7–9 slices each were harvested after 6 days of treatment, sonicated, and equal protein aliquots assessed for the 20S proteasome α-1 subunit (d), synaptophysin (SNP; e), and actin. Mean immunoreactivity levels were normalized to their respective controls and percent ± SEM values are shown. Unpaired t-tests: ***p< 0.001.</p

Among screened compounds, PADK was chosen as an effective CatB-enhancing agent for further testing in the hippocampal slice model.

<p>Among screened compounds, PADK was chosen as an effective CatB-enhancing agent for further testing in the hippocampal slice model.</p

Lactacystin-induced proteasome inhibition leads to the enhancement of CatB activity.

<p>Lactacystin-induced proteasome inhibition leads to the enhancement of CatB activity.</p

Aβ₄₂-induced proteasome inhibition is associated with a delayed, inverse effect on CatB activity.

<p>Hippocampal slice cultures were treated daily with vehicle for 6 days (0-day control group) or with pre-aggregated Aβ₄₂ for 4–6 days, staggering the treatments in order for same-day harvesting of slice groups of 7–9 each. Proteasome activity (gray plot of mean Vmax/s ± SEM; ANOVA: p<0.01) and cathepsin B activity (black plot of mean fluorometric units ± SEM; ANOVA: p<0.01) were measured in equal protein aliquots from the same samples (a). The time-course samples were also assessed for 55–70-kDa pTau-Ser^199/202 and actin in order to plot the within-sample ratios between the immunoreactivity levels (b; mean ± SEM). Tukey post hoc tests compared to vehicle-treated slices: **p<0.01, ***p<0.001.</p

The effective CatB-enhancing agent PADK (chosen from Table 2) selectively enhances CatB activity in hippocampal slice cultures.

<p>The slice cultures were treated daily with vehicle or 3 μM PADK for 2 days before being collected into slice groups of 7–9 each. Panel a: Immunoblot assessments stained the 30- and 25-kDa CatB isoform (CatB-30 and CatB-25), GluR1, and synaptophysin (SNP). Panel b: Fluorogenic peptide assays assessed the harvested slice samples for cathepsin B and proteasome chymotrypsin-like activities. The two measures were normalized to their respective vehicle control groups (mean ± SEM). Cathepsin B activity exhibited a significant increase, whereas proteasome activity exhibited only a small increase. Panel c: Percent changes in CatB (white) and proteasome (black) activities compared to control are shown for 6-day Aβ₄₂ treatment (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182895#pone.0182895.g003" target="_blank">Fig 3a</a> data), for 4-day lactacystin treatment (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182895#pone.0182895.g004" target="_blank">Fig 4a</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182895#pone.0182895.t001" target="_blank">Table 1</a> data), and for 2-day PADK treatment (from Fig 7b data).</p