Florian M. Gebhardt

Glutamate transporter variants reduce glutamate uptake in Alzheimer's disease

A characteristic of Alzheimers disease (AD) is that neuron populations in the temporal, frontal, and parietal cortices are selectively vulnerable. Several neurotransmitters have been proposed to play roles in neural destruction as AD progresses, including glutamate. Failure to clear the synaptic cleft of glutamate can overstimulate postsynaptic glutamate receptors, promoting neuronal death. Excitatory amino acid transporter 2 (EAAT2), which is concentrated in perisynaptic astrocytes, performs 90% of glutamate uptake in mammalian central nervous system. Alternative splicing of EAAT2 mRNA could regulate glutamate transport in normal and disease states. We report disease- and pathology-specific variations in EAAT2 splice variant expression in AD brain obtained at autopsy. While wild type EAAT2 showed a global reduction in expression, brain regions susceptible to neuronal loss demonstrated greater expression of transcripts that reduced glutamate transport in an in vitro assay. Functional splice variant EAAT2b showed no significant variation with disease state. These results have implications for the treatment of AD as modulators of EAAT2 splicing and/or glutamate uptake would augment current therapies aimed at blocking glutamate receptors.

Exon-skipping Splice Variants of Excitatory Amino Acid Transporter-2 (EAAT2) Form Heteromeric Complexes with Full-length EAAT2

The glial transporter excitatory amino acid transporter-2 (EAAT2) is the main mediator of glutamate clearance in brain. The wild-type transporter (EAAT2wt) forms trimeric membrane complexes in which each protomer functions autonomously. Several EAAT2 variants are found in control and Alzheimer-diseased human brains; their expression increases with pathological severity. These variants might alter EAAT2wt-mediated transport by abrogating membrane trafficking, or by changing the configuration or functionality of the assembled transporter complex. HEK293 cells were transfected with EAAT2wt; EAAT2b, a C-terminal variant; or either of two exon-skipping variants: alone or in combination. Surface biotinylation studies showed that only the exon-7 deletion variant was not trafficked to the membrane when transfected alone, and that all variants could reach the membrane when co-transfected with EAAT2wt. Fluorescence resonance energy transfer (FRET) studies showed that co-transfected EAAT2wt and EAAT2 splice variants were expressed in close proximity. Glutamate transporter function was measured using a whole cell patch clamp technique, or by changes in membrane potential indexed by a voltage-sensitive fluorescent dye (FMP assay): the two methods gave comparable results. Cells transfected with EAAT2wt or EAAT2b showed glutamate-dependent membrane potential changes consistent with functional expression. Cells transfected with EAAT2 exon-skipping variants alone gave no response to glutamate. Co-transfection of EAAT2wt (or EAAT2b) and splice variants in various ratios significantly raised glutamate EC50 and decreased Hill coefficients. We conclude that exon-skipping variants form heteromeric complexes with EAAT2wt or EAAT2b that traffic to the membrane but show reduced glutamate-dependent activity. This could allow glutamate to accumulate extracellularly and promote excitotoxicity.

Housekeepers for accurate transcript expression analysis in Alzheimer's disease autopsy brain tissue

Quantitative real‐time reverse transcription–polymerase chain reaction (qRT‐PCR) is a popular technique for mRNA expression studies. Normalization to an endogenous reference transcript (housekeeper) is widely used to correct for differences in loading and RNA quality. Alzheimers disease (AD) alters brain metabolism. The stability of housekeeper transcript expression must be carefully validated.

Analysis of multiple exon-skipping mRNA splice variants using SYBR Green real-time RT-PCR

Fluorescence-based PCR techniques are becoming an increasingly popular method for measuring low-abundance alternatively spliced mRNA transcripts. The dynamic range of real-time RT-PCR affords high sensitivity for the measurement of gene expression, but this mandates the need for strict controls to ensure assay validity. Primer design, reverse transcription, and cycling conditions need to be optimized to ensure an accurate and reproducible assay. Here, we describe a procedure for creating a cost effective and reliable method for the absolute quantification of several exon-skipping variants of human excitatory amino acid transporter-2 (EAAT2). We show that the cycling conditions can be adjusted to increase the specificity of primers that span exon-exon junctions, and that differences in the reverse transcription reaction can be minimized. Standard curves are stable and produce accurate absolute copy number data. We report that exon-skipping transcripts, EAAT2Delta7 and EAAT2Delta9, account for 5.8% of EAAT2 mRNA in autopsy human neocortex.

Metabolic Abnormalities in Alzheimer Disease

In 1906, German neuropathologist and psychiatrist Alois Alzheimer described “eine eigenartige Erkrankung der Hirnrinde” (a peculiar disease of the cerebral cortex). Alzheimer noted two abnormalities in autopsied brain tissue from his index case: senile plaques, proteinaceous structures previously described in the brain of normal elderly people; and abnormal cells delineated with silver stain that became known as neurofibrillary tangles (NFTs). The distribution and abundance of tangle-filled neurons are now the main criteria used to diagnose Alzheimer disease (AD) at autopsy.

Exon-skipping variants of excitatory amino acid transporter-2 in Alzheimer's disease: correlation with APOE genotype

Astrocytes are remarkably multifunctional cells. Not only do astrocytes function as the primary supportive cell type of brain, but they may actively regulate synaptic transmission by release of gliotransmitters. The phenotype of astrocytes are changed in cultures or in acute brain slices and these preparations may not be ideal to define the roles of astrocytes. Furthermore, a major challenge for the future is to define the role of astrocytes in complex tasks, such as sensory processing and functional hyperemia, which is not feasible ex vivo. In vivo imaging of astrocytes in barrel cortex using 2-photon laser scanning microscopy represents a powerful new approach to define how astrocytes contribute to both normal physiological processing and to the evolution of acute and degenerative neurological diseases. Imaging of NADH can be use as an intrinsic indicator of cellular redox state in cortex using 2-photon excitation. Fluorescent calcium indicators can be loaded selectively into astrocytes and combined with recording of multi-unit activity for monitoring of neural activity and labeling of plasma with FITC-dextran to study astrocytic response to sensory input (whisker stimulation). Photolysis of caged calcium represents a selective and non-invasive approach to stimulate single astrocytes or their vascular processes. Use of transgenic mice expressing GFP or its spectral variants under cell specific promoters is ideally suited to analyze the interaction of astrocytes with other cells types in cortex. Rodent models of ischemia, epilepsy, and subarachnoid bleeding suggest that astrocytes are activated at early stages in each of these diseases and that astrocytes at multiple steps are involved in their pathogenesis. PL1 In vivo imaging of astrocytes in adult brain

Real-time quantitative RT-PCR analysis of EAAT2 splice variants

Apolipoprotein E4 (apoE4) is a risk factor for the sporadic form of Alzheimer’s disease (AD). In addition, some clinical studies indicate that high doses of vitamin E may be useful in the treatment of AD. In this study we examined the modulation of brain vitamin E concentrations by apoE. ApoE deficient (knockout) and control mice were placed on normal rodent chow and the tocopherol concentrations in different regions of brains determined by HPLC. At age 2.5 months (initial age) all regions of the apoE deficient brains contained less alpha tocopherol than the corresponding regions in controls. This trend continued for 9.5 months for most CNS areas except the spinal cord and cerebellum. For example, cerebellar alpha tocopherol in apoE deficient animals was 71 ± 2.5% of controls initially and increased to 103 ± 2.4 % at the end of the study. It is postulated that the unusual cellular architecture and metabolism of cerebellum, especially the astrocytic cells such as the Bergmann glia, may play a role in the unique behavior of the cerebellum. Alpha tocopherol and cholesterol were high in the sera of the apoE deficient animals. However, unlike tocopherol, brain cholesterol levels were the same in the deficient and control mice. This suggests that a) the decline in brain alpha tocopherol in apoE deficiency is not due to general alterations in lipid metabolism and b) the processing of alpha tocopherol in brain follows a separate pathway than that of cholesterol. The data suggest that apoE exerts some control over the uptake of vitamin E across the blood brain barrier and/or it’s uptake, retention and metabolism in brain. The effects of apoE on vitamin E processing were different among the various regions of the brain. Supported by research funds from the Veterans Administration.