Christensen, Trace

AbstractBackgroundAlzheimer’s disease (AD) is an age‐dependent neurodegenerative disorder with limited treatment options. As it progresses, synapse degeneration is the most important feature contributing to cognitive dysfunction. Mitochondria supply synapses with ATP for neurotransmitter release and vesicle recycling and buffer calcium concentrations. In AD, mitochondrial function is disrupted leading to energetic stress and synapse degeneration. We have identified a novel therapeutic strategy that blocks neurodegeneration and protects cognitive function in multiple mouse models of AD (amyloid and tau pathologies) via mild pharmacological inhibition of mitochondria complex I, which results in the activation of adaptive stress response and subsequent improvement of mitochondrial function.MethodAnalysis of mitochondrial morphology and synaptic density was done in the hippocampal CA1 tissue from APP/PS1 and non‐transgenic (NTG) mice treated with vehicle or mild complex I inhibitor CP2 for 14 months starting after the development of cognitive dysfunction and neurodegeneration. A serial block‐face scanning electron microscopy (SBF‐SEM) and three‐dimensional (3D) EM reconstructions were employed to determine the synaptic density and mitochondrial morphology and distribution associated with different dendritic spine types.ResultWe demonstrate that APP/PS1 mice have significantly reduced synaptic density in the hippocampus compared to the NTG littermates. CP2 treatment restores synaptic density to the levels observed in NTG mice evident by the increase in the number of synaptic densities visible in the electron micrographs. Mitochondrial morphology and association with synapses are currently being investigated.ConclusionOur data are consistent with previously conducted electrophysiology recording in acute brain slices from APP/PS1 and NTG mice treated with vehicle or CP2, demonstrating restoration of long‐term potentiation (LTP) after chronic CP2 treatment. Additionally, it suggests that 3D EM‐based assessment of synaptic density could serve as a surrogate marker for LTP and cognitive function useful for drug discovery.

ABSTRACT Phage therapy has not been established in the clinical routine, in part due to uncertainties concerning efficacy and immunogenicity. Here, three rabbits were immunized against staphylococcal phage K to assess viral potency in the presence of immunized serum. Three rabbits received weekly intramuscular injections of ~10 10±1 pfu/mL phage K. Phage K-specific IgG formation was measured by an enzyme-linked immunosorbent assay (ELISA); phage inactivation was assessed by calculating K-rates. Using transmission electron microscopy (TEM) and immunogold labeling, antibody binding to phage K was visualized. This was numerically assessed by objective imaging analysis comparing the relative distances of each gold particle to the nearest phage head and tail structure. Immunization led to a strong IgG response, plateauing 7 days after the last phage injection. There was no significant correlation between K-rate and antibody titer over time. TEM showed IgG binding to the head structure of phage K. Image analysis showed a significant reduction in relative distances between antibodies and phage head structures when comparing samples from day 0 and day 28 ( P < 0.0001). These results suggest that while individual serum analysis for antibodies against therapeutic phage bears consideration prior to and with prolonged therapy, during phage application, the formation of specific antibodies against phage may only partially explain decreased phage potency in the presence of immunized serum. Instead, other factors may contribute to an individual’s “humoral receptiveness” to phage therapy. Future investigations should be directed toward the identification of the humoral factors that have the most significant predictive value on phage potency in vivo .

AbstractBackgroundAlzheimer’s Disease (AD) has no cure. We showed that partial inhibition of mitochondrial complex I (MCI) with small molecule CP2 induced adaptive stress response activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD.MethodsAnalysis of mitochondrial morphology and mitochondria‐endoplasmic reticulum (ER) contacts (MERCS) was done in the hippocampal CA1 tissue from APP/PS1 mice treated with CP2 and compared to APP/PS1 and non‐transgenic mice treated with vehicle using serial block‐face scanning electron microscopy (SBFSEM) and three‐dimensional (3D) EM reconstructions combined with western blot analysis and next generation RNA sequencing.ResultsWe demonstrate that CP2 treatment restores mitochondrial morphology and mitochondria‐ ER communication, reducing ER stress in the APP/PS1 mouse brain. Using 3D EM reconstructions, we found that mitochondria in AD dendrites existed primarily as mitochondria‐on‐a‐string (MOAS), a morphological phenotype that is difficult to detect using conventional 2D EM or fluorescence spectroscopy. Compared to other morphological phenotypes, MOAS are extensively enveloped in the ER membranes forming multiple MERCS known to contribute to abnormal lipid and calcium homeostasis, and ER stress in AD. CP2 treatment specifically reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reduction in MERCS, ER stress, and improved lipid homeostasis.ConclusionsThese data provide novel information on the role MOAS play in AD. Our data demonstrate that in response to energetic stress, mitochondria in dendrites acquire MOAS phenotype with increased interaction with the ER, which may synergistically contribute to the AD development. Since CP2 treatment alleviated the ER stress and improved mitochondrial dynamics and function, these data further support the development of partial MCI inhibitors as disease modifying strategy for AD.

Abstract During aging, muscle gradually undergoes sarcopenia, the loss of function associated with loss of mass, strength, endurance, and oxidative capacity. However, the 3D structural alterations of mitochondria associated with aging in skeletal muscle and cardiac tissues are not well described. Although mitochondrial aging is associated with decreased mitochondrial capacity, the genes responsible for the morphological changes in mitochondria during aging are poorly characterized. We measured changes in mitochondrial morphology in aged murine gastrocnemius, soleus, and cardiac tissues using serial block‐face scanning electron microscopy and 3D reconstructions. We also used reverse transcriptase‐quantitative PCR, transmission electron microscopy quantification, Seahorse analysis, and metabolomics and lipidomics to measure changes in mitochondrial morphology and function after loss of mitochondria contact site and cristae organizing system (MICOS) complex genes, Chchd3 , Chchd6 , and Mitofilin . We identified significant changes in mitochondrial size in aged murine gastrocnemius, soleus, and cardiac tissues. We found that both age‐related loss of the MICOS complex and knockouts of MICOS genes in mice altered mitochondrial morphology. Given the critical role of mitochondria in maintaining cellular metabolism, we characterized the metabolomes and lipidomes of young and aged mouse tissues, which showed profound alterations consistent with changes in membrane integrity, supporting our observations of age‐related changes in muscle tissues. We found a relationship between changes in the MICOS complex and aging. Thus, it is important to understand the mechanisms that underlie the tissue‐dependent 3D mitochondrial phenotypic changes that occur in aging and the evolutionary conservation of these mechanisms between Drosophila and mammals.

This article shows how mitochondria in murine cardiac changes, importantly elucidating age-related changes. It also is the first to show that the MICOS complex may play a role in outer membrane mitochondrial structure.

In the original publication [...]

Abstract Mitochondria respond to metabolic demands of the cell and to incremental damage, in part, through dynamic structural changes that include fission (fragmentation), fusion (merging of distinct mitochondria), autophagic degradation (mitophagy), and biogenic interactions with the endoplasmic reticulum (ER). High resolution study of mitochondrial structural and functional relationships requires rapid preservation of specimens to reduce technical artifacts coupled with quantitative assessment of mitochondrial architecture. A practical approach for assessing mitochondrial fine structure using two dimensional and three dimensional high‐resolution electron microscopy is presented, and a systematic approach to measure mitochondrial architecture, including volume, length, hyperbranching, cristae morphology, and the number and extent of interaction with the ER is described. These methods are used to assess mitochondrial architecture in cells and tissue with high energy demand, including skeletal muscle cells, mouse brain tissue, and Drosophila muscles. The accuracy of assessment is validated in cells and tissue with deletion of genes involved in mitochondrial dynamics.

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa −/− null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa −/− zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa −/− mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa −/− mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa −/− mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.