Identification of a BACE dimer and characterization of its biochemical and enzymatic properties

The deposition amyloid β peptide in the brains of patients is a hallmark of Alzheimer’s disease and is thought to play a major pathogenetic role in the development of the demential symptoms of this severe illness. The amyloid β peptide is generated from the β-amyloid precursor protein (APP) by cleavage of the “β-site APP-cleaving enzyme” (BACE) followed by cleavage of the "gamma-secretase”. Whereas it has recently been discovered that the gamma-secretase is a multi-protein complex, it has not yet been investigated whether under native conditions, BACE functions in association with other proteins. The present work thus studied BACE by means of blue native gel electrophoresis and found that native BACE has a molecular weight of 140 kDa, whereas BACE under denaturing conditions has a molecular weight of 70 kDa which is only half of its native mass. Co-immunoprecipitation experiments with differently tagged full-length BACE constructs subsequently showed that this higher molecular weight species of BACE corresponds to a BACE homodimer. In contrast, a BACE ectodomain, lacking the C-terminus and the transmembrane domain, is a monomer. A consecutive domain analysis revealed that both the C-terminus and the transmembrane domain of BACE are dispensable for dimerization. In line with this, it could be shown that the ectodomain of BACE can dimerize if it is attached to the membrane by a GPI anchor. In terms of the cellular localization of the dimerization process, it could furthermore be demonstrated that retention of BACE in the ER by addition of a KKXX-motif does not prevent dimerization. This suggests that dimerization can occur prior to full maturation of BACE which takes place in the Golgi apparatus. In addition, kinetic analyses of the purified native BACE dimer revealed a higher affinity and turnover rate for an APP-like substrate in comparison to the monomeric soluble BACE ectodomain. This suggests a putative function of dimerization in improving enzymatic efficiency. The implication of these findings for our understanding of the Amyloid-β synthesis as well as for a putatively alternative therapeutic strategy are discussed

Photoacoustic Neuroimaging - Perspectives on a Maturing Imaging Technique and its Applications in Neuroscience

A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain’s network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective. Here, we thus present the latest advances in biomedical and biological engineering on photoacoustic neuroimaging in the context of complementary imaging techniques. A particular focus is placed on recent advances in whole-brain photoacoustic imaging in rodent models and its influential role in bridging the gap between fluorescence microscopy and more non-invasive techniques such as magnetic resonance imaging (MRI). We consider current strategies to address persistent challenges, particularly in developing molecular contrast agents, and conclude with an overview of potential future directions for photoacoustic neuroimaging to provide deeper insights into healthy and pathological brain processes

A reversible state of hypometabolism in a human cellular model of sporadic Parkinson's disease

Sporadic Parkinson's Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the alpha-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD. Mitochondrial dysfunction is a contributing factor in Parkinson's disease. Here the authors carry out a multilayered omics analysis of Parkinson's disease patient-derived neuronal cells, which reveals a reversible hypometabolism mediated by alpha-ketoglutarate dehydrogenase deficiency, which is correlated with disease progression in the donating patients

Reward magnitude tracking by neural populations in ventral striatum

Evaluation of the magnitudes of intrinsically rewarding stimuli is essential for assigning value and guiding behavior. By combining parametric manipulation of a primary reward, medial forebrain bundle (MFB) microstimulation, with functional magnetic imaging (fMRI) in rodents, we delineated a broad network of structures activated by behaviorally characterized levels of rewarding stimulation. Correlation of psychometric behavioral measurements with fMRI response magnitudes revealed regions whose activity corresponded closely to the subjective magnitude of rewards. The largest and most reliable focus of reward magnitude tracking was observed in the shell region of the nucleus accumbens (NAc). Although the nonlinear nature of neurovascular coupling complicates interpretation of fMRI findings in precise neurophysiological terms, reward magnitude tracking was not observed in vascular compartments and could not be explained by saturation of region-specific hemodynamic responses. In addition, local pharmacological inactivation of NAc changed the profile of animals’ responses to rewards of different magnitudes without altering mean reward response rates, further supporting a hypothesis that neural population activity in this region contributes to assessment of reward magnitudes.United States. Defense Advanced Research Projects Agency (Grant W911NF-10-0059)National Institutes of Health (U.S.) (Grant R01-DA028299)National Institutes of Health (U.S.) (Grant R01-DA038642

A reversible state of hypometabolism in a human cellular model of sporadic Parkinson’s disease

Abstract Sporadic Parkinson’s Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD

Non-invasive and high-throughput interrogation of exon-specific isoform expression

Truong et al. developed a cell-based reporter system, EXSISERS, that enables non-invasive quantification of the protein expression levels of exon-specific isoforms via intein-mediated protein splicing. Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions