Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

Background Danger associated molecular patterns (DAMPs) are nuclear or cytoplasmic proteins that are released from the injured tissues and activate the innate immune system. Mitochondrial DNA (mtDNA) is a novel DAMP that is released into the extracellular milieu subsequent to cell death and injury. We hypothesized that cell death within the central nervous system in children with traumatic brain injury (TBI) would lead to release of mtDNA into the cerebrospinal fluid (CSF) and has the potential to predict the outcome after trauma. Methods CSF was collected from children with severe TBI that required intracranial pressure monitoring with Glasgow Coma Scale (GCS) scores ≤ 8 via an externalized ventricular drain. Control CSF was obtained in children without TBI or meningoencephalitis that demonstrated no leukocytes in the diagnostic lumbar puncture. Results The median age for patients with TBI was 6.3 y and 62% were male. The common mechanisms of injury included motor vehicle collision (35.8%) followed by falls (21.5%) and inflicted TBI (19%); 6 children (14.2%) died during their ICU course. The mean CSF mtDNA concentration was 1.10E +05 ± 2.07E+05 and 1.63E+03 ± 1.80E+03 copies/µL in the pediatric TBI and control population respectively. Furthermore, the mean CSF mtDNA concentration in pediatric patients who later died or had severe disability was significantly higher than that of the survivors (1.63E+ 05 ± 2.77E+05 vs. 5.05E+04 ± 6.21E+04 copies/µL) (p<0.0001). We found a significant correlation between CSF mtDNA and HMGB1, another prototypical DAMP, concentrations (ρ = 0.574, p<0.05), supporting the notion that both DAMPs are increased in the CSF following TBI. Conclusions Our data suggest that CSF mtDNA is novel DAMP in TBI, and appears to be a useful biomarker that correlates with neurological outcome after TBI. Further inquiry into the components of mtDNA that modulate the innate immune response will be helpful in understanding the mechanism of local and systemic inflammation after TBI.

Mitochondrial DNA (mtDNA) is a novel danger-associated molecular pattern that on its release into the extracellular milieu acts via toll-like receptor-9, a pattern recognition receptor of the immune system. We hypothesized that plasma mtDNA concentrations will be elevated in septic children, and these elevations are associated with an increase in the severity of illness. In a separate set of in vitro experiments, we test the hypothesis that exposing peripheral blood mononuclear cells (PBMC) to mtDNA activates the immune response and induces tumor necrosis factor (TNF) release. Children with sepsis/systemic inflammatory response syndrome or control groups were enrolled within 24 h of admission to the pediatric intensive care unit. Mitochondrial gene cytochrome c oxidase 1 (COX1) concentrations were measured by realtime quantitative PCR in the DNA extracted from plasma. PBMCs were treated with mtDNA (10μg/mL) and supernatant TNF levels were measured. The median plasma mtDNA concentrations were significantly elevated in the septic patients as compared with the critically ill non-septic and healthy control patients [1.75E+05 (IQR 6.64E+04-3.67E+05) versus 5.73E+03 (IQR 3.90E+03-1.28E+04) and 6.64E+03 (IQR 5.22E+03-1.63E+04) copies/μL respectively]. The median concentrations of plasma mtDNA were significantly greater in patients with MOF as compared with patients without MOF (3.2E+05 (IQR 1.41E+05-1.08E+06) vs. 2.9E+04 (IQR 2.47E+04-5.43E+04) copies/μL). PBMCs treated with mtDNA demonstrated higher supernatant TNF levels as compared with control cells (6.5 ±1.8 vs. 3.5±0.5 pg/mL, P>0.05). Our data suggest that plasma mtDNA is a novel danger-associated molecular pattern in pediatric sepsis and appears to be associated with MOF.