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)

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

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 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

One at a time, live tracking of NGF axonal transport using quantum dots

Retrograde axonal transport of nerve growth factor (NGF) signals is critical for the survival, differentiation, and maintenance of peripheral sympathetic and sensory neurons and basal forebrain cholinergic neurons. However, the mechanisms by which the NGF signal is propagated from the axon terminal to the cell body are yet to be fully elucidated. To gain insight into the mechanisms, we used quantum dot-labeled NGF (QD-NGF) to track the movement of NGF in real time in compartmentalized culture of rat dorsal root ganglion (DRG) neurons. Our studies showed that active transport of NGF within the axons was characterized by rapid, unidirectional movements interrupted by frequent pauses. Almost all movements were retrograde, but short-distance anterograde movements were occasionally observed. Surprisingly, quantitative analysis at the single molecule level demonstrated that the majority of NGF-containing endosomes contained only a single NGF dimer. Electron microscopic analysis of axonal vesicles carrying QD-NGF confirmed this finding. The majority of QD-NGF was found to localize in vesicles 50–150 nm in diameter with a single lumen and no visible intralumenal membranous components. Our findings point to the possibility that a single NGF dimer is sufficient to sustain signaling during retrograde axonal transport to the cell body

Altered Neurocircuitry in the Dopamine Transporter Knockout Mouse Brain

The plasma membrane transporters for the monoamine neurotransmitters dopamine, serotonin, and norepinephrine modulate the dynamics of these monoamine neurotransmitters. Thus, activity of these transporters has significant consequences for monoamine activity throughout the brain and for a number of neurological and psychiatric disorders. Gene knockout (KO) mice that reduce or eliminate expression of each of these monoamine transporters have provided a wealth of new information about the function of these proteins at molecular, physiological and behavioral levels. In the present work we use the unique properties of magnetic resonance imaging (MRI) to probe the effects of altered dopaminergic dynamics on meso-scale neuronal circuitry and overall brain morphology, since changes at these levels of organization might help to account for some of the extensive pharmacological and behavioral differences observed in dopamine transporter (DAT) KO mice. Despite the smaller size of these animals, voxel-wise statistical comparison of high resolution structural MR images indicated little morphological change as a consequence of DAT KO. Likewise, proton magnetic resonance spectra recorded in the striatum indicated no significant changes in detectable metabolite concentrations between DAT KO and wild-type (WT) mice. In contrast, alterations in the circuitry from the prefrontal cortex to the mesocortical limbic system, an important brain component intimately tied to function of mesolimbic/mesocortical dopamine reward pathways, were revealed by manganese-enhanced MRI (MEMRI). Analysis of co-registered MEMRI images taken over the 26 hours after introduction of Mn^(2+) into the prefrontal cortex indicated that DAT KO mice have a truncated Mn^(2+) distribution within this circuitry with little accumulation beyond the thalamus or contralateral to the injection site. By contrast, WT littermates exhibit Mn^(2+) transport into more posterior midbrain nuclei and contralateral mesolimbic structures at 26 hr post-injection. Thus, DAT KO mice appear, at this level of anatomic resolution, to have preserved cortico-striatal-thalamic connectivity but diminished robustness of reward-modulating circuitry distal to the thalamus. This is in contradistinction to the state of this circuitry in serotonin transporter KO mice where we observed more robust connectivity in more posterior brain regions using methods identical to those employed here