Luteolin Reduces Alzheimer’s Disease Pathologies Induced by Traumatic Brain Injury

Traumatic brain injury (TBI) occurs in response to an acute insult to the head and is recognized as a major risk factor for Alzheimer’s disease (AD). Indeed, recent studies have suggested a pathological overlap between TBI and AD, with both conditions exhibiting amyloid-beta (Aβ) deposits, tauopathy, and neuroinflammation. Additional studies involving animal models of AD indicate that some AD-related genotypic determinants may be critical factors enhancing temporal and phenotypic symptoms of TBI. Thus in the present study, we examined sub-acute effects of moderate TBI delivered by a gas-driven shock tube device in Aβ depositing Tg2576 mice. Three days later, significant increases in b-amyloid deposition, glycogen synthase-3 (GSK-3) activation, phospho-tau, and pro-inflammatory cytokines were observed. Importantly, peripheral treatment with the naturally occurring flavonoid, luteolin, significantly abolished these accelerated pathologies. This study lays the groundwork for a safe and natural compound that could prevent or treat TBI with minimal or no deleterious side effects in combat personnel and others at risk or who have experienced TBI

Anti-human α-synuclein N-terminal peptide antibody protects against dopaminergic cell death and ameliorates behavioral deficits in an AAV-α-synuclein rat model of Parkinson's disease.

The protein α-synuclein (α-Syn) has a central role in the pathogenesis of Parkinson's disease (PD) and immunotherapeutic approaches targeting this molecule have shown promising results. In this study, novel antibodies were generated against specific peptides from full length human α-Syn and evaluated for effectiveness in ameliorating α-Syn-induced cell death and behavioral deficits in an AAV-α-Syn expressing rat model of PD. Fisher 344 rats were injected with rAAV vector into the right substantia nigra (SN), while control rats received an AAV vector expressing green fluorescent protein (GFP). Beginning one week after injection of the AAV-α-Syn vectors, rats were treated intraperitoneally with either control IgG or antibodies against the N-terminal (AB1), or central region (AB2) of α-Syn. An unbiased stereological estimation of TH+, NeuN+, and OX6 (MHC-II) immunostaining revealed that the α-Syn peptide antibodies (AB1 and AB2) significantly inhibited α-Syn-induced dopaminergic cell (DA) and NeuN+ cell loss (one-way ANOVA (F (3, 30) = 5.8, p = 0.002 and (F (3, 29) = 7.92, p = 0.002 respectively), as well as decreasing the number of activated microglia in the ipsilateral SN (one-way ANOVA F = 14.09; p = 0.0003). Antibody treated animals also had lower levels of α-Syn in the ipsilateral SN (one-way ANOVA F (7, 37) = 9.786; p = 0.0001) and demonstrated a partial intermediate improvement of the behavioral deficits. Our data suggest that, in particular, an α-Syn peptide antibody against the N-terminal region of the protein can protect against DA neuron loss and, to some extent behavioral deficits. As such, these results may be a potential therapeutic strategy for halting the progression of PD

Effect of intraperitoneal administered anti-α-Syn antibodies on the number of OX-6+ cells (MHCII).

<p>Micrographs of anti-OX6 staining for (A) AAV-GFP, (B) AAV-α-Syn + IgG, (C) AAV-α-Syn + AB1, and (D) AAV-α-Syn + AB2. Strong immunoreactivity for OX-6 is shown in the inset at high power magnification (40X). Increased OX-6 immunoreactivity was present in AAV-α-Syn + IgG treated rats compared to the other three groups. (E) Graph of unbiased stereological estimation of OX6+ cells in the SN. There is a significant decreased in the number of OX-6+ cells in groups that received anti-α-Syn antibodies (AB1,AB2) compared with the control IgG groups. Data are shown as mean ± SEM. (AAV-GFP control [n = 14], AAV α-Syn + IgG [n = 9], AB1 [n = 8], and AB2 [n = 6] were analyzed) ***P< 0.0001 ** P< 0.001 by one-way ANOVA with post-hoc Bonferroni test. Scale bar = 100μm.</p

Study design to test the efficacy of anti- α-Syn antibody in rat AAV- α-Syn PD model.

<p>A schematic diagram depicting (A) the details on AAV9 concentrations used and the time frame for AAV-9 injections and the behavioral testing, (B) the timing of the first antibody injections and initial dose, as indicated in the methods section the dose of the AB injected was reduced over time (C) the sequence of the antibodies and (D) level of serum antibodies taken 1 week after injections and just before subsequent injections. This demonstrates that antibody levels remained high for the first month and then clearance increased after 6 weeks. Times of injections are indicated by the arrows on the bottom of the graph.</p

Effect of intraperitoneal administered anti-α-Syn antibodies on motor function.

<p>AAV-α-Syn+IgG rats demonstrated paw bias in the cylinder test when compared with AAV-GFP controls starting at two months and continued at 3 months, likely reflecting the progressive nature of this model with ongoing DA cell loss (A two-way ANOVA found a main effect of treatment, F_3,67 = 4.48, p = <0.001). Although AB1 or AB2 antibody treatment did not demonstrate any significant improvement in behavioral deficits compared to AAV-α-Syn+IgG treated animals, at no time was the AB1 nor AB2 group significantly different from the control AAV-GFP group. Data are presented, as the percent right paw preference ± SEM (n = 23 AAV-GFP control and n = 16 for treatment groups).</p

The effect of intraperitoneal administered anti-α-Syn antibodies on AAV vector mediated α-Syn expression.

<p>Immunostaining of the SN region with an antibody against α-Syn. Administration of AAV-α-Syn into the rat SN caused significant expression of α-Syn in the SN (B) compared to the AAV-GFP control group (A). Intraperitoneal injection of anti-α-Syn antibody AB1 reduced α-Syn level in the SN (C), while injection with antibody AB2 had a reduced effect (D). Quantitative analysis of levels of α-Syn expression is presented as percent positive area (E). Data are presented as the percent positive area of anti-α-Syn staining throughout the SN (n = 8 animals per group). Asterisk denotes significance (*** P<0.001, * P<0.05) with comparison made to the ipsilateral AAV-GFP group by 1-way ANOVA with post-hoc Bonferroni test. ELISA analysis confirmed a significant reduction in α-Syn levels in the SN with antibody AB1 compared to IgG treatment (F). ### P< 0.001, # P< 0.05 vs. Control AAV-α-Syn + IgG. Data are presented as the mean concentration of α-Syn in pg/μg of protein ± SEM (n = 6 per group). Scale bars are 100μm.</p

Rescue of TH+ and NeuN+ cells in the ipsilateral SN with intraperitoneal administration of anti-α-Syn antibodies.

<p>Immunohistochemical staining of the SN region with an anti-TH antibody (A) AAV-GFP, (B) AAV-α-Syn + IgG, (C) AAV-α-Syn + AB1, (D) AAV-α-Syn + AB2. (E) Graph of unbiased stereological estimation of TH+ cells in the SN of treated animals. AB1 treated animals showed similar levels of TH+ cells compared to the GFP control and significantly higher number of TH+ cells compared to the IgG treated group. Data are shown as mean ± SEM (n = 13 AAV-GFP control and n = 7 for treatment groups). (F) Graph of NeuN+ cells of the SN. Stereologic analysis shows a significant rescue of NeuN+ cells in SN sections with AB1 compared with IgG treated animals (n = 9 AAV-GFP control and n = 7 for treatment groups). *P< 0.05, **P< 0.01, ***P< 0.001. Scale bar = 100μm.</p

Paw preference for all groups.

<p>Paw preference for all groups.</p