Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca2+-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol. © 1982

Automated Computational Processing of 3-D MR Images of Mouse Brain for Phenotyping of Living Animals

Magnetic resonance (MR) imaging provides a method to obtain anatomical information from the brain in vivo that is not typically available by optical imaging because of this organ's opacity. MR is nondestructive and obtains deep tissue contrast with 100-µm^3 voxel resolution or better. Manganese-enhanced MRI (MEMRI) may be used to observe axonal transport and localized neural activity in the living rodent and avian brain. Such enhancement enables researchers to investigate differences in functional circuitry or neuronal activity in images of brains of different animals. Moreover, once MR images of a number of animals are aligned into a single matrix, statistical analysis can be done comparing MR intensities between different multi-animal cohorts comprising individuals from different mouse strains or different transgenic animals, or at different time points after an experimental manipulation. Although preprocessing steps for such comparisons (including skull stripping and alignment) are automated for human imaging, no such automated processing has previously been readily available for mouse or other widely used experimental animals, and most investigators use in-house custom processing. This protocol describes a stepwise method to perform such preprocessing for mouse

Decoupling the effect of mutant amyloid precursor protein (APP) from the effect of plaque on axonal transport dynamics in the living mouse brain: A correlation MRI-microscopy study

The parent protein for amyloid plaques, amyloid precursor protein (APP), mediates cargo‐motor attachments for intracellular transport. Axonal transport is decreased and the distal location of accumulation is altered in transgenic mice expressing human APP with the Swedish and Indiana mutations (APPSwInd) linked to Familial Alzheimer’s Disease, as detected by time‐lapse magnetic resonance imaging (MRI) of transport in living mouse brains (Bearer et al. 2017). Transport is also altered in brains of Down syndrome mice with 3 copies of APP gene. Questions now become whether expression of mutated APP effects transport dynamics independent of plaque, and do plaques alone contribute to transport defects? To address these we used the Tet‐Off system to decouple expression of APPSwInd from presence of plaques, and then studied transport using our MRI technique in three experimental groups of transgenic mice in which the timing and duration of APPSwInd expression, and thereby plaque formation, was altered with doxycycline: Group A (+ plaques, + APPSwInd); Group B (+ plaques, no APPSwInd), and group C (no plaques, + APPSwInd). Manganese‐enhanced MRI (MEMRI) allows us to perform cell biological experiments in live animals with T1‐weighted MRI in a Bruker 11.7T scanner (Medina et al 2016). Time‐lapse MR images were captured before and after stereotactic injection of Mn2+ (3‐5nL) into CA3 of the hippocampus at successive time‐points. Images of multiple individuals were aligned and processed with our automated computational pipeline (Medina et al. 2017) and statistical parametric mapping (SPM) performed. After MRI brains were harvested for histopathology or biochemistry. Results show that within group between time‐point have altered transport locations as well as diminished transport in all groups compared to wildtype (p<0.05 FDR n= 36). Preliminary ANOVA between‐group comparisons both by SPM and by region of interest measurements of images support the visual impression that APPSwInd expression alone may compromise transport. Groups A and B displayed plaques, but not C, and Western blots showed APPSwInd expressed 3.2‐fold over normal at sacrifice in Groups A and C but not B, with Aβ detected only in Groups A and B, where phospho‐tau was also present in dystrophic neurites surrounding plaques. Cholinergic neurons that project to hippocampus from the medial septal nucleus were decreased in Group C (p=0.0006 by ANOVA, n=15). Isolated hippocampal vesicles contained Mn2+, as well as Trk (NGF receptor), Rab 5 and 7 (associated with transport vesicles), suggesting a distinct vesicle population is affected by these APP mutations. These surprising results implicate mutated APPSwInd in transport defects, separable from the effect of plaque

Developing a Model for Slow Hypoxic Injury and Vascular Degeneration in Amyloid Burdened Brains

The breakdown of neurovascular systems may play a crucial role in the pathogenesis of Alzheimer’s disease. However whether this breakdown initiates a degenerative mechanism or is the consequence of some other deleterious process remains unknown. We examined hippocampal pathology in double transgenic mice overexpressing a human mutant gene encoding the amyloid precursor protein (APPSwe/Ind) using a combination of histochemistry and stereologic techniques. Expression of APPSwe/Ind in these mice is driven by a tetracycline-sensitive promoter. Tetracycline transcriptional activator (tTA), the second transgene, is driven in turn by a CAM KIIa promoter that is only active in neurons. Thus this double transgenic construct allows us to control expression of APPSwe/Ind with doxycycline. Utilizing this characteristic, we created three distinct experimental groups: A, display abeta plaque pathology and express APPSwe/Ind at time of sacrifice; B, display abeta plaque pathology but do not express APPSwe/Ind at time of sacrifice; and C, do not display abeta plaque pathology but do express APPSwe/Ind at time of sacrifice. Stereologic investigation revealed decreased hippocampal volume in groups A(n=5) and B(n=5) when compared to group C(n=5) and age-matched wildtype (n=9)

Witnessing microtubule-based transport in the living brain: Impact of the cargomotor receptor, amyloid precursor protein, and Alzheimer’s plaques

Most amyloid precursor protein (APP)-based Alzheimer’s models overexpress mutant human APP resulting in Abeta plaques. Yet the relative contribution of this elevated APP and the presence of plaques to neurodegeneration remains a big question. APP’s role as a cargo-motor receptor for axonal transport suggests that overexpression might lead to increased transport. Indeed we showed that transport is increased in Down’s syndrome and decreased in APP knockout mice. Hence transport may be elevated in APP overexpressors and lead to either beneficial or deleterious consequences. Here we use high field microMRI with Mn2+, an MR contrast agent useful as a track-tracer, to pose this cell biological quest ion within the whole living brains of wildtype and Alzheimer’s model mice. Injection of Mn2+ into the CA3 region of the hippocampus results in measurable transport over time. Application of 3D unbiased whole brain image analysis detects all circuitry emanating from the hippocampus. By driving APP Swe/Ind transgene expression with a tetracycline-sensitive promoter, APPSwe/Ind expression can be decoupled from the presence of plaques with doxycycline (doxy). Three groups of mice were studied: group ‘A’ (no doxy, +plaques, +APP); group ‘B’ (doxy at 8 days before sacrifice, +plaques, no APP), and group ‘C’ (doxy prior to conception, and stopped 8 days before sacrifice, no plaques, +APP). Images were captured before and sequentionally after Mn2+ injection into CA 3 (1, 7, 25 hr). Images were aligned and analyzed by statistical parametric mapping to identify differential accumulation within the hippocampal projections. Histopathology revealed well-developed plaques in A and B, and Western blots showed human APP expressed five-fold over WT in in A and C. Our preliminary results show increased transport in A and C, with APP Swe/Ind expression when compared with B, where expression is suppressed. Cholinergic neurons in the medial septal nucleus were decreased as determined by anti-ChAT staining in Group C (p=0.0006 by one-way ANOVA, n=15). In conclusion, the effects of elevated APP expression are separable from consequences of plaque, and each may

Decoupling the Effects of the Amyloid Precursor Protein From Amyloid-β Plaques on Axonal Transport Dynamics in the Living Brain

Amyloid precursor protein (APP) is the precursor to Aβ plaques. The cytoplasmic domain of APP mediates attachment of vesicles to molecular motors for axonal transport. In APP-KO mice, transport of Mn²⁺ is decreased. In old transgenic mice expressing mutated human (APP^(SwInd)) linked to Familial Alzheimer’s Disease, with both expression of APP^(SwInd) and plaques, the rate and destination of Mn²⁺ axonal transport is altered, as detected by time-lapse manganese-enhanced magnetic resonance imaging (MEMRI) of the brain in living mice. To determine the relative contribution of expression of APP^(SwInd) versus plaque on transport dynamics, we developed a Tet-off system to decouple expression of APP^(SwInd) from plaque, and then studied hippocampal to forebrain transport by MEMRI. Three groups of mice were compared to wild-type (WT): Mice with plaque and APP^(SwInd) expression; mice with plaque but suppression of APP^(SwInd) expression; and mice with APP^(SwInd) suppressed from mating until 2 weeks before imaging with no plaque. MR images were captured before at successive time points after stereotactic injection of Mn²⁺ (3–5 nL) into CA3 of the hippocampus. Mice were returned to their home cage between imaging sessions so that transport would occur in the awake freely moving animal. Images of multiple mice from the three groups (suppressed or expressed) together with C57/B6J WT were aligned and processed with our automated computational pipeline, and voxel-wise statistical parametric mapping (SPM) performed. At the conclusion of MR imaging, brains were harvested for biochemistry or histopathology. Paired T-tests within-group between time points (p = 0.01 FDR corrected) support the impression that both plaque alone and APP^(SwInd) expression alone alter transport rates and destination of Mn²⁺ accumulation. Expression of APP^(SwInd) in the absence of plaque or detectable Aβ also resulted in transport defects as well as pathology of hippocampus and medial septum, suggesting two sources of pathology occur in familial Alzheimer’s disease, from toxic mutant protein as well as plaque. Alternatively mice with plaque without APP^(SwInd) expression resemble the human condition of sporadic Alzheimer’s, and had better transport. Thus, these mice with APP^(SwInd) expression suppressed after plaque formation will be most useful in preclinical trials

A simple rapid process for semi-automated brain extraction from magnetic resonance images of the whole mouse head

Background: Magnetic resonance imaging (MRI) is a well-developed technique in neuroscience. Limitations in applying MRI to rodent models of neuropsychiatric disorders include the large number of animals required to achieve statistical significance, and the paucity of automation tools for the critical early step in processing, brain extraction, which prepares brain images for alignment and voxel-wise statistics. New Method: This novel timesaving automation of template-based brain extraction (“skull-stripping”) is capable of quickly and reliably extracting the brain from large numbers of whole head images in a single step. The method is simple to install and requires minimal user interaction. Results: This method is equally applicable to different types of MR images. Results were evaluated with Dice and Jacquard similarity indices and compared in 3D surface projections with other stripping approaches. Statistical comparisons demonstrate that individual variation of brain volumes are preserved. Comparison with Existing Methods: A downloadable software package not otherwise available for extraction of brains from whole head images is included here. This software tool increases speed, can be used with an atlas or a template from within the dataset, and produces masks that need little further refinement. Conclusions: Our new automation can be applied to any MR dataset, since the starting point is a template mask generated specifically for that dataset. The method reliably and rapidly extracts brain images from whole head images, rendering them useable for subsequent analytical processing. This software tool will accelerate the exploitation of mouse models for the investigation of human brain disorders by MRI

Decoupling the effect of mutant amyloid precursor protein (APP) from the effect of plaque on axonal transport dynamics in the living mouse brain: A correlation MRI-microscopy study

The parent protein for amyloid plaques, amyloid precursor protein (APP), mediates cargo‐motor attachments for intracellular transport. Axonal transport is decreased and the distal location of accumulation is altered in transgenic mice expressing human APP with the Swedish and Indiana mutations (APPSwInd) linked to Familial Alzheimer’s Disease, as detected by time‐lapse magnetic resonance imaging (MRI) of transport in living mouse brains (Bearer et al. 2017). Transport is also altered in brains of Down syndrome mice with 3 copies of APP gene. Questions now become whether expression of mutated APP effects transport dynamics independent of plaque, and do plaques alone contribute to transport defects? To address these we used the Tet‐Off system to decouple expression of APPSwInd from presence of plaques, and then studied transport using our MRI technique in three experimental groups of transgenic mice in which the timing and duration of APPSwInd expression, and thereby plaque formation, was altered with doxycycline: Group A (+ plaques, + APPSwInd); Group B (+ plaques, no APPSwInd), and group C (no plaques, + APPSwInd). Manganese‐enhanced MRI (MEMRI) allows us to perform cell biological experiments in live animals with T1‐weighted MRI in a Bruker 11.7T scanner (Medina et al 2016). Time‐lapse MR images were captured before and after stereotactic injection of Mn2+ (3‐5nL) into CA3 of the hippocampus at successive time‐points. Images of multiple individuals were aligned and processed with our automated computational pipeline (Medina et al. 2017) and statistical parametric mapping (SPM) performed. After MRI brains were harvested for histopathology or biochemistry. Results show that within group between time‐point have altered transport locations as well as diminished transport in all groups compared to wildtype (p<0.05 FDR n= 36). Preliminary ANOVA between‐group comparisons both by SPM and by region of interest measurements of images support the visual impression that APPSwInd expression alone may compromise transport. Groups A and B displayed plaques, but not C, and Western blots showed APPSwInd expressed 3.2‐fold over normal at sacrifice in Groups A and C but not B, with Aβ detected only in Groups A and B, where phospho‐tau was also present in dystrophic neurites surrounding plaques. Cholinergic neurons that project to hippocampus from the medial septal nucleus were decreased in Group C (p=0.0006 by ANOVA, n=15). Isolated hippocampal vesicles contained Mn2+, as well as Trk (NGF receptor), Rab 5 and 7 (associated with transport vesicles), suggesting a distinct vesicle population is affected by these APP mutations. These surprising results implicate mutated APPSwInd in transport defects, separable from the effect of plaque