Alzheimer's disease (AD) is a disease of aging that results in cognitive impairment, dementia, and death. Pathognomonic features of AD are amyloid plaques composed of proteolytic fragments of the amyloid precursor protein (APP) and neurofibrillary tangles composed of hyperphosphorylated tau protein. One type of familial AD occurs when mutant forms of APP are inherited. Both APP and tau are components of the microtubule-based axonal transport system, which prompts the hypothesis that axonal transport is disrupted in AD, and that such disruption impacts cognitive function. Transgenic mice expressing mutated forms of APP provide preclinical experimental systems to study AD. Here, we perform manganese-enhanced magnetic resonance imaging to study transport from hippocampus to forebrain in four cohorts of living mice: young and old wild-type and transgenic mice expressing a mutant APP with both Swedish and Indiana mutations (APPSwInd). We find that transport is decreased in normal aging and further altered in aged APPSwInd plaque-bearing mice. These findings support the hypothesis that transport deficits are a component of AD pathology and thus may contribute to cognitive deficits.
Magnetic resonance (MR) imaging provides a method to obtain anatomical information from the brain in vivo not amenable to optical imaging because of its opacity. MR is non-destructive and obtains deep tissue contrast with 100 µm3 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, comprised of individuals from different mouse strains, different transgenic animals, or between 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 step-wise method to perform such preprocessing for mouse.
Microtubule-based motors carry cargo back and forth between the synaptic region and the cell body. Defects in axonal transport result in peripheral neuropathies, some of which are caused by mutations in KIF5A, a gene encoding one of the heavy chain isoforms of conventional kinesin-1. Some mutations in KIF5A also cause severe central nervous system defects in humans. While transport dynamics in the peripheral nervous system have been well characterized experimentally, transport in the central nervous system is less experimentally accessible and until now not well described. Here we apply manganese-enhanced magnetic resonance (MEMRI) to study transport dynamics within the central nervous system, focusing on the hippocampal-forebrain circuit, and comparing kinesin-1 light chain 1 knock-out (KLC-KO) mice with age-matched wild-type littermates. We injected Mn2+ into CA3 of the posterior hippocampus and imaged axonal transport in vivo by capturing whole-brain 3D magnetic resonance images (MRI) in living mice at discrete time-points after injection. Precise placement of the injection site was monitored in both MR images and in histologic sections. Mn2+-induced intensity progressed along fiber tracts (fimbria and fornix) in both genotypes to the medial septal nuclei (MSN), correlating in location with the traditional histologic tract tracer, rhodamine dextran. Pairwise statistical parametric mapping (SPM) comparing intensities at successive time-points within genotype revealed Mn2+-enhanced MR signal as it proceeded from the injection site into the forebrain, the expected projection from CA3. By region of interest (ROI) analysis of the MSN, wide variation between individuals in each genotype was found. Despite this statistically significant intensity increases in the MSN at 6 hr post-injection was found in both genotypes, albeit less so in the KLC-KO. While the average accumulation at 6 hr was less in the KLC-KO, this difference did not reach significance. Projections of SPM T-maps for each genotype onto the same grayscale image revealed differences in the anatomical location of significant voxels. Although KLC-KO mice had smaller brains than wild-type, the gross anatomy was normal with no apparent loss of septal cholinergic neurons. Hence anatomy alone does not explain the differences in SPM maps. We conclude that kinesin-1 defects may have only a minor effect on the rate and distribution of transported Mn2+ within the living brain. This impairment is less than expected for this abundant microtubule-based motor, yet such defects could still be functionally significant, resulting in cognitive/emotional dysfunction due to decreased replenishments of synaptic vesicles or mitochondria during synaptic activity. This study demonstrates the power of MEMRI to observe and measure vesicular transport dynamics in the central nervous system that may result from or lead to brain pathology.
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
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 (APPSwInd) linked to Familial Alzheimer’s Disease, with both expression of APPSwInd 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 APPSwInd versus plaque on transport dynamics, we developed a Tet-off system to decouple expression of APPSwInd 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 APPSwInd expression; mice with plaque but suppression of APPSwInd expression; and mice with APPSwInd 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 APPSwInd expression alone alter transport rates and destination of Mn2+ accumulation. Expression of APPSwInd 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 APPSwInd expression resemble the human condition of sporadic Alzheimer’s, and had better transport. Thus, these mice with APPSwInd expression suppressed after plaque formation will be most useful in preclinical trials.
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