Dendritic vulnerability in neurodegenerative disease: insights from analyses of cortical pyramidal neurons in transgenic mouse models

Abstract In neurodegenerative disorders, such as Alzheimer's disease, neuronal dendrites and dendritic spines undergo significant pathological changes. Because of the determinant role of these highly dynamic structures in signaling by individual neurons and ultimately in the functionality of neuronal networks that mediate cognitive functions, a detailed understanding of these changes is of paramount importance. Mutant murine models, such as the Tg2576 APP mutant mouse and the rTg4510 tau mutant mouse have been developed to provide insight into pathogenesis involving the abnormal production and aggregation of amyloid and tau proteins, because of the key role that these proteins play in neurodegenerative disease. This review showcases the multidimensional approach taken by our collaborative group to increase understanding of pathological mechanisms in neurodegenerative disease using these mouse models. This approach includes analyses of empirical 3D morphological and electrophysiological data acquired from frontal cortical pyramidal neurons using confocal laser scanning microscopy and whole-cell patchclamp recording techniques, combined with computational modeling methodologies. These collaborative studies are designed to shed insight on the repercussions of dystrophic changes in neocortical neurons, define the cellular phenotype of differential neuronal vulnerability in relevant models of neurodegenerative disease, and provide a basis upon which to develop meaningful therapeutic strategies aimed at preventing, reversing, or compensating for neurodegenerative changes in dementia

Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication

<p>Abstract</p> <p>Background</p> <p>SHANK3 is a protein in the core of the postsynaptic density (PSD) and has a critical role in recruiting many key functional elements to the PSD and to the synapse, including components of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA), metabotropic glutamate (mGlu) and <it>N</it>-methyl-D-aspartic acid (NMDA) glutamate receptors, as well as cytoskeletal elements. Loss of a functional copy of the <it>SHANK3 </it>gene leads to the neurobehavioral manifestations of 22q13 deletion syndrome and/or to autism spectrum disorders. The goal of this study was to examine the effects of haploinsufficiency of full-length <it>Shank3 </it>in mice, focusing on synaptic development, transmission and plasticity, as well as on social behaviors, as a model for understanding <it>SHANK3 </it>haploinsufficiency in humans.</p> <p>Methods</p> <p>We used mice with a targeted disruption of <it>Shank3 </it>in which exons coding for the ankyrin repeat domain were deleted and expression of full-length Shank3 was disrupted. We studied synaptic transmission and plasticity by multiple methods, including patch-clamp whole cell recording, two-photon time-lapse imaging and extracellular recordings of field excitatory postsynaptic potentials. We also studied the density of GluR1-immunoreactive puncta in the CA1 stratum radiatum and carried out assessments of social behaviors.</p> <p>Results</p> <p>In <it>Shank3 </it>heterozygous mice, there was reduced amplitude of miniature excitatory postsynaptic currents from hippocampal CA1 pyramidal neurons and the input-output (I/O) relationship at Schaffer collateral-CA1 synapses in acute hippocampal slices was significantly depressed; both of these findings indicate a reduction in basal neurotransmission. Studies with specific inhibitors demonstrated that the decrease in basal transmission reflected reduced AMPA receptor-mediated transmission. This was further supported by the observation of reduced numbers of GluR1-immunoreactive puncta in the stratum radiatum. Long-term potentiation (LTP), induced either with θ-burst pairing (TBP) or high-frequency stimulation, was impaired in <it>Shank3 </it>heterozygous mice, with no significant change in long-term depression (LTD). In concordance with the LTP results, persistent expansion of spines was observed in control mice after TBP-induced LTP; however, only transient spine expansion was observed in <it>Shank3 </it>heterozygous mice. Male <it>Shank3 </it>heterozygotes displayed less social sniffing and emitted fewer ultrasonic vocalizations during interactions with estrus female mice, as compared to wild-type littermate controls.</p> <p>Conclusions</p> <p>We documented specific deficits in synaptic function and plasticity, along with reduced reciprocal social interactions in <it>Shank3 </it>heterozygous mice. Our results are consistent with altered synaptic development and function in <it>Shank3 </it>haploinsufficiency, highlighting the importance of Shank3 in synaptic function and supporting a link between deficits in synapse function and neurodevelopmental disorders. The reduced glutamatergic transmission that we observed in the <it>Shank3 </it>heterozygous mice represents an interesting therapeutic target in <it>Shank3</it>-haploinsufficiency syndromes.</p

Dietary composition modulates brain mass and solubilizable Aβ levels in a mouse model of aggressive Alzheimer's amyloid pathology

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Objective Alzheimer's disease (AD) is a progressive neurodegenerative disease of the central nervous system (CNS). Recently, an increased interest in the role diet plays in the pathology of AD has resulted in a focus on the detrimental effects of diets high in cholesterol and fat and the beneficial effects of caloric restriction. The current study examines how dietary composition modulates cerebral amyloidosis and neuronal integrity in the TgCRND8 mouse model of AD. Methods From 4 wks until 18 wks of age, male and female TgCRND8 mice were maintained on one of four diets: (1) reference (regular) commercial chow; (2) high fat/low carbohydrate custom chow (60 kcal% fat/30 kcal% protein/10 kcal% carbohydrate); (3) high protein/low carbohydrate custom chow (60 kcal% protein/30 kcal% fat/10 kcal% carbohydrate); or (4) high carbohydrate/low fat custom chow (60 kcal% carbohydrate/30 kcal% protein/10 kcal% fat). At age 18 wks, mice were sacrificed, and brains studied for (a) wet weight; (b) solubilizable A&#946; content by ELISA; (c) amyloid plaque burden; (d) stereologic analysis of selected hippocampal subregions. Results Animals receiving a high fat diet showed increased brain levels of solubilizable A&#946;, although we detected no effect on plaque burden. Unexpectedly, brains of mice fed a high protein/low carbohydrate diet were 5% lower in weight than brains from all other mice. In an effort to identify regions that might link loss of brain mass to cognitive function, we studied neuronal density and volume in hippocampal subregions. Neuronal density and volume in the hippocampal CA3 region of TgCRND8 mice tended to be lower in TgCRND8 mice receiving the high protein/low carbohydrate diet than in those receiving the regular chow. Neuronal density and volume were preserved in CA1 and in the dentate gyrus. Interpretation Dissociation of A&#946; changes from brain mass changes raises the possibility that diet plays a role not only in modulating amyloidosis but also in modulating neuronal vulnerability. However, in the absence of a study of the effects of a high protein/low carbohydrate diet on nontransgenic mice, one cannot be certain how much, if any, of the loss of brain mass exhibited by high protein/low carbohydrate diet-fed TgCRND8 mice was due to an interaction between cerebral amyloidosis and diet. Given the recent evidence that certain factors favor the maintenance of cognitive function in the face of substantial structural neuropathology, we propose that there might also exist factors that sensitize brain neurons to some forms of neurotoxicity, including, perhaps, amyloid neurotoxicity. Identification of these factors could help reconcile the poor clinicopathological correlation between cognitive status and structural neuropathology, including amyloid pathology.Published versio

Amyloid Triggers Extensive Cerebral Angiogenesis Causing Blood Brain Barrier Permeability and Hypervascularity in Alzheimer's Disease

Evidence of reduced blood-brain barrier (BBB) integrity preceding other Alzheimer's disease (AD) pathology provides a strong link between cerebrovascular angiopathy and AD. However, the “Vascular hypothesis”, holds that BBB leakiness in AD is likely due to hypoxia and neuroinflammation leading to vascular deterioration and apoptosis. We propose an alternative hypothesis: amyloidogenesis promotes extensive neoangiogenesis leading to increased vascular permeability and subsequent hypervascularization in AD. Cerebrovascular integrity was characterized in Tg2576 AD model mice that overexpress the human amyloid precursor protein (APP) containing the double missense mutations, APPsw, found in a Swedish family, that causes early-onset AD. The expression of tight junction (TJ) proteins, occludin and ZO-1, were examined in conjunction with markers of apoptosis and angiogenesis. In aged Tg2576 AD mice, a significant increase in the incidence of disrupted TJs, compared to age matched wild-type littermates and young mice of both genotypes, was directly linked to an increased microvascular density but not apoptosis, which strongly supports amyloidogenic triggered hypervascularity as the basis for BBB disruption. Hypervascularity in human patients was corroborated in a comparison of postmortem brain tissues from AD and controls. Our results demonstrate that amylodogenesis mediates BBB disruption and leakiness through promoting neoangiogenesis and hypervascularity, resulting in the redistribution of TJs that maintain the barrier and thus, provides a new paradigm for integrating vascular remodeling with the pathophysiology observed in AD. Thus the extensive angiogenesis identified in AD brain, exhibits parallels to the neovascularity evident in the pathophysiology of other diseases such as age-related macular degeneration

Automated Three-Dimensional Detection and Shape Classification of Dendritic Spines from Fluorescence Microscopy Images

A fundamental challenge in understanding how dendritic spine morphology controls learning and memory has been quantifying three-dimensional (3D) spine shapes with sufficient precision to distinguish morphologic types, and sufficient throughput for robust statistical analysis. The necessity to analyze large volumetric data sets accurately, efficiently, and in true 3D has been a major bottleneck in deriving reliable relationships between altered neuronal function and changes in spine morphology. We introduce a novel system for automated detection, shape analysis and classification of dendritic spines from laser scanning microscopy (LSM) images that directly addresses these limitations. The system is more accurate, and at least an order of magnitude faster, than existing technologies. By operating fully in 3D the algorithm resolves spines that are undetectable with standard two-dimensional (2D) tools. Adaptive local thresholding, voxel clustering and Rayburst Sampling generate a profile of diameter estimates used to classify spines into morphologic types, while minimizing optical smear and quantization artifacts. The technique opens new horizons on the objective evaluation of spine changes with synaptic plasticity, normal development and aging, and with neurodegenerative disorders that impair cognitive function

The role of inflammation and amyloid beta in Alzheimer disease pathology

Alzheimer disease (AD) is the most common form of dementia. Due to longer life-spans the number of affected individuals is expected to triple over the next few decades. As a consequence, a great deal of research is focused on determining the many processes by which the disease manifests as well as in discovering biomarkers and therapeutics to aid in diagnosis and disease prevention. The neuropathological hallmarks of AD include extracellular deposits of amyloid into senile plaques, accumulation of abnormal Tau filaments into neurofibrillary tangles, extensive neurodegeneration and inflammation. Although significant advances have been made in AD neurodegeneration, there still remain many unanswered and unforeseen aspects to the disease. It has been established that microglia, the immune cells of the brain, become activated in response to amyloid; however, the precise intracellular responses of microglia to amyloid and the relationship between microglia and amyloid deposition or clearance is unresolved. There have been many genes identified whose expression is upregulated in activated microglia and many of them have been proposed to be used as markers for inflammation. It has been demonstrated in humans that serum levels of melanotransferrin (p97), an iron binding molecule, is elevated in individuals affected with AD and that it is the activated, plaque-associated microglia that are responsible for this upregulation. This thesis further investigated the association between microglial activation and p97 gene expression and found that the levels of p97, both mRNA and protein, are increased in activated microglia in culture. The change in gene expression occurred largely in response to amyloid treatment possibly by the regulation of the AP-1 transcription factor downstream of the p38 mitogen-activated protein kinase pathway.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Aged Tg2576 Mice have reduced tight function expression.

<p>The expression of occludin or ZO-1 were compared quantitatively between wild-type and Tg2576 mice in the neocortex (A and B) and hippocampus (C and D). (A) The percentage of cortical cerebral blood vessels with abnormal ZO-1 expression patterns was significantly higher in the aged Tg2576 mice compared to age-matched wild-type (***p<0.001). The incidence of ZO-1 disruption was also significantly higher in aged Tg2576 mice compared to young Tg2576 (Young wild-type, n = 4; Young Tg2576, n = 3; aged wild-type, n = 5; aged Tg2576, n = 4; *p<0.05) in the cortex. (B) Aged Tg2576 mice had significantly reduced occludin protein levels in the cortex compared to age-matched wild-type (n = 7, **p = 0.0072). (C) The percentage of hippocampal cerebral blood vessels with abnormal ZO-1 expression patterns was significantly higher in the aged Tg2576 mice compared to age-matched wild-type (***p<0.001). Similarly, the incidence of ZO-1 disruption was also significantly higher in aged Tg2576 mice compared to young Tg2576 (Young wild-type, n = 4; Young Tg2576, n = 3; aged wild-type, n = 5; aged Tg2576, n = 4; *p<0.05) in the hippocampus. (D) Aged Tg2576 mice had significantly reduced occludin protein levels in the hippocampus compared to age-matched wild-type (n = 7, **p = 0.0076). Values represent mean ± SEM.</p

Angiogenesis not apoptosis induces alterations in tight junction immunoreactivity in Tg2576 mice.

<p>Representative confocal micrographs of TJs (ZO-1), double stained for markers of angiogenesis or apoptosis in aged wild-type and Tg2576 mice. All vessels stained for CD105 regardless of the TJ expression pattern. White arrowheads point to regions of TJ abnormality in the vasculature. Double staining of blood vessels with ZO-1 (red) and CD105 (green) in wild-type (A). and Tg2576 (B) neocortex. Double staining of vessels with ZO-1 (red) and caspase 3 (green) in wild-type (C) and Tg2576 (D) neocortex. Caspase-3 staining did not colocalize with ZO-1 staining indicating an absence of apoptosis in the vasculature. Results are representative of three separate experiments of three mice per group of brain tissues examined. Scale bar represents 20 µm.</p