Aβ Vaccination in Combination with Behavioral Enrichment in Aged Beagles: Effects on Cognition, Aβ, and Microhemorrhages

Beta-amyloid (Aβ) immunotherapy is a promising intervention to slow Alzheimer’s disease (AD). Aging dogs naturally accumulate Aβ and show cognitive decline. An active vaccine against fibrillar Aβ 1–42 (VAC) in aged beagles resulted in maintenance but not improvement of cognition along with reduced brain Aβ. Behavioral enrichment (ENR) led to cognitive benefits but no reduction in Aβ. We hypothesized cognitive outcomes could be improved by combining VAC with ENR in aged dogs. Aged dogs (11–12 years) were placed into 4 groups: (1) control/control (C/C); (2) control/VAC (C/V); (3) ENR/control (E/C); (4) ENR and VAC (E/V) and treated for 20 months. VAC decreased brain Aβ, pyroglutamate Aβ, increased CSF Aβ42 and BDNF RNA levels but also increased microhemorrhages. ENR reduced brain Aβ and prevented microhemorrhages. The combination treatment resulted in a significant maintenance of learning over time, reduced Aβ and increased BDNF mRNA despite increased microhemorrhages, however there were no benefits to memory. These results suggest that the combination of immunotherapy with behavioral enrichment leads to cognitive maintenance associated with reduced neuropathology that may benefit people with AD

Sensing Intracellular Calcium: Stim1 And Orai1 Interactions At The Plasma Membrane

Store operated Ca2+ entry (SOCE) is a ubiquitous process in nonexcitable cells important for receptor signaling in hematopoietic and other cell types. The ERtransmembrane Ca2+ sensor STIM1 and the plasma membrane Ca2+ channel Orai1 are the two essential protein components of SOCE. To further understand the mechanism by which STIM1 and Orai1 facilitate SOCE, I have characterized bimolecular and supramolecular electrostatic interactions involving these proteins, using an imagingbased fluorescence resonance energy transfer (FRET) assay in RBL mast cells. Using this assay I initially identified positively-charged small molecule inhibitors of the STIM1-Orai1 interaction. Based on this information I hypothesized that these small molecules could be binding to and disrupting the C-terminal acidic coiled-coil of Orai1. Mutation of these acidic residues in Orai1 reduced its association with STIM1 and caused constitutive clustering of Orai1 at the plasma membrane. I then identified a short polybasic sequence in the Ca2+ activating domain (CAD) of STIM1 that binds to this acidic region of Orai1. Mutation of this three amino acid basic sequence prevented association with wild type Orai1, but not with the Orai1 coiled-coil mutant. Despite the residual association between the Orai1 and STIM1 mutants, they cannot initiate SOCE, suggesting that the polybasic sequence in STIM1 and the acidic coiled-coil of Orai1 are important for Ca2+ gating. Using multiple isoforms of type I phosphoinositide-5-kinase (PI5KI) and lipid domain targeted inositol-5-phosphatases, I found that the STIM1-Orai1 interaction has a dual dependence on two pools of of phosphoinositide-(4,5)-bisphosphate (PIP2) in the plasma membrane that are distinguishable by their association with detergent resistant membranes and detergent solubilized membranes. These correspond to distinctive ordered lipid subregions and disordered lipid subregions in the membrane, respectively. Deletion of an N-terminal polyarginine sequence on Orai1 or a Cterminal polylysine sequence on STIM1 interferes with these selective interactions with PIP2 pools localized to these subregions. Based on these findings, I propose a model in which Orai1 must translocate between functionally distinct membrane domains in a PIP2 dependent fashion, to engage STIM1 associated with PIP2 in ordered lipid subregions of the membrane

Synaptic UNC13A protein variant causes increased neurotransmission and dyskinetic movement disorder

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies