SNCA genetic lowering reveals differential cognitive function of alpha-synuclein dependent on sex

Antisense oligonucleotide (ASO) therapy for neurological disease has been successful in clinical settings and its potential has generated hope for Alzheimer's disease (AD). We previously described that ablating SNCA encoding for alpha-synuclein (alpha Syn) in a mouse model of AD was beneficial. Here, we sought to demonstrate whether transient reduction of alpha Syn expression using ASO(SNCA) could be therapeutic in a mouse model of AD. The efficacy of the ASO(SNCA) was measured via immunocytochemistry, RT-qPCR and western blotting. To assess spatial learning and memory, ASO(SNCA) or PBS-injected APP and non-transgenic (NTG) mice, and separate groups of SNCA-null mice, were tested on the Barnes circular maze. Hippocampal slice electrophysiology and transcriptomic profiling were used to explore synaptic function and differential gene expression between groups. Reduction of SNCA transcripts alleviated cognitive deficits in male transgenic animals, but surprisingly, not in females. To determine the functional cause of this differential effect, we assessed memory function in SNCA-null mice. Learning and memory were intact in male mice but impaired in female animals, revealing that the role of alpha Syn on cognitive function is sex-specific. Transcriptional analyses identified a differentially expressed gene network centered around EGR1, a central modulator of learning and memory, in the hippocampi of SNCA-null mice. Thus, these novel results demonstrate that the function of alpha Syn on memory differs between male and female brains.This work was supported by grants from the National Institutes of Health (NIH) to SEL (RF1-AG044342, RF1-AG070296, R21-AG065693, R01-AG077743, R01-NS092918, R01-AG062135 and R56-NS113549), to MKL (AG062135, NS108686, NS086074, NS092093). Training grant support for graduate students (T32-NS105604). This study was supported by a grant from the Winston and Maxine Wallin Neuroscience Discovery Fund. Additional support included start-up funds from the University of Minnesota Foundation and bridge funds from the Institute of Translational Neuroscience to SEL

The apoptotic response in HCT116BAX-/- cancer cells becomes rapidly saturated with increasing expression of a GFP-BAX fusion protein

Abstract Background Many chemotherapeutic agents promote tumor cell death by activating the intrinsic pathway of apoptosis. Intrinsic apoptosis involves permeabilization of the mitochondrial outer membrane and the release of cytochrome c, a process that is controlled by proteins of the BCL2 gene family. Chemoresistance is often associated with abnormalities in concentrations of BCL2 family proteins. Although stoichiometirc interactions between anti-apoptotic and BH3-only BCL2 family proteins have been well documented as affecting cell death, the association between changes in BAX concentration and intrinsic apoptosis are poorly understood. Methods Exogenous GFP-murine Bax fusion constructs were transfected into BAX-deficient HCT116 cells. To titrate the expression of the fusion protein, GFP-BAX was cloned into a tetracycline sensitive expression cassette and cotransfected with a plasmid expressing the rtTA transcription factor into HCT116 BAX-/- cells. Linear expression of the fusion gene was induced with doxycycline and monitored by quantitative PCR and immunoblotting. Cell death was assayed by DAPI staining cells after exposure to indomethacin, and scoring nuclei for condensed chromatin and fragmented nuclei. Results HCT116 BAX-/- cells were resistant to indomethacin, but susceptibility could be recovered in cells expressing a GFP-BAX fusion protein. Titration of GFP-BAX expression revealed that the concentration of BAX required to induce a saturating apoptosis response from baseline, was rapidly achieved. Increased levels of GFP-BAX were unable to stimulate higher levels of cell death. Examination of GFP-BAX distribution before and after indomethacin treatment indicated that BAX protein did not form aggregates when present at sub-lethal concentrations. Conclusion Within the limitations of this experimental system, BAX-dependent apoptosis in HCT116 cells exhibits an all-or-none response depending on the level of BAX protein present. The lack of BAX aggregation at sub-saturation levels suggests that the translocation step of BAX activation may be impaired

Patent Foramen Ovale: Standards for a Preclinical Model of Prevalence, Structure, and Histopathologic Comparability to Human Hearts

BACKGROUND: This study evaluated and standardized a Patent Foramen Ovale (PFO) preclinical model in gross anatomic and histopathologic features. METHODS: We examined 150 necropsy-derived domestic porcine hearts, age 4-6 months for PFO prevalence, appearance, and size. Histopathologic preparations were standardized and processed identically to 24 post-mortem human hearts aged 16-62 years. A measurement scheme was developed for PFO atrial openings, tunnel length, and histopathologic features to compare porcine and patient hearts. RESULTS: PFO was found in 32 of the 150 porcine hearts (prevalence 21.3%). Twenty-five porcine PFO underwent standard characterization by tunnel length, and right, and left atrial orifice diameters. Logarithmic regression analysis between porcine PFO tunnel length and left atrial orifice area demonstrated a significant positive relationship (P = 0.0162, R(2) = 0.227). The porcine PFO tunnel length was significantly longer than in humans (12.0 +/- 4.0 mm vs. 7.1 +/- 3.1 mm respectively, P \u3c 0.0001). Histopathologic comparison was made using serial sections perpendicular to the atrial septum and the tunnel long axis. Human and porcine PFO lesions demonstrated strong similarities in tissue cells, connective tissue, and matrix composition. CONCLUSIONS: PFO assessment was standardized in both macroscopically and histopathologically, with quantitative and qualitative comparisons feasible using a porcine preclinical model. PFO prevalence in domestic swine is identical to humans, and microscopic structures very similar to humans. The domestic swine PFO model appears useful to evaluate new interventional closure technologies due to comparability in microscopic features. Tunnel length should be carefully evaluated due to differences across pigs and patients