Paradoxical upregulation of glutamatergic presynaptic boutons during mild cognitive impairment

Abstract

Synaptic integrity is now recognized as a central component of Alzheimer's disease. Surprisingly, however, the structural status of glutamatergic synapses in Alzheimer's disease is unclear, despite the fact that glutamate is the major excitatory transmitter of the CNS and has key roles in excitotoxicity and long-term potentiation. The identification of specific markers of glutamatergic neurons now allows an assessment of the structural involvement of the glutamatergic system across progressive stages of the Alzheimer's pathology, an opportunity not afforded by previously used neurochemical approaches. Glutamatergic presynaptic bouton density and dystrophic neurite abundance were quantified in midfrontal gyrus brain tissue from subjects with no cognitive impairment, mild cognitive impairment, or mild- or severe-stage Alzheimer's disease. Our study demonstrates a striking pathology-dependent pattern of glutamatergic synaptic remodeling with disease progression. Subjects with mild cognitive impairment display a paradoxical elevation in glutamatergic presynaptic bouton density, a situation akin to that observed in the cholinergic system, which then depletes and drops with disease progression. This pattern of synaptic remodeling mirrors our previous findings in transgenic animal models and is of major relevance to current transmitter-based therapeutics.

Figure 1.

Immunohistochemical staining of the glutamatergic presynaptic bouton sites in human midfrontal gyrus tissue using antibodies directed against the glutamatergic presynaptic bouton site-specific marker vesicular glutamate transporter 1. A–D, Stainings are from subjects with no cognitive impairment (A), mild cognitive impairment (B), mild AD (C), or severe AD (D). Note the elevation in terminal number in B, taken from a subject with mild cognitive impairment, and the decreased presynaptic bouton-immunoreactivities in C and D, taken from subjects with mild and severe AD, respectively. E–G, The quantification protocol used to determine glutamatergic presynaptic bouton density in the midfrontal gyrus. Original digital images as shown in E were transformed into a file type that increases the computer's accuracy of detection (F). This transformed file format is then quantified by the computer using precise inclusion and exclusion criteria (as described in Materials and Methods) to accurately detect the elements of interest. G, The quantified image, in which dark blue coloration indicates those elements that were quantified, and light blue coloration indicates elements that failed to meet the required criteria and were hence omitted. Quantified data are then tallied and yielded in number format. Scale bars: D (for A–D), G (for E–G), 10 μm.

Figure 2.

Graphic depiction of the relationship between glutamatergic presynaptic bouton density (number of presynaptic boutons per 1000 μm² area) and cognitive ability. A, Mean midfrontal gyrus glutamatergic presynaptic bouton densities in subjects grouped by cognitive ability (NCI, MCI, mAD, and sAD). Means were compared by ANOVA followed by post hoc Tukey's test. B–E, Positive associations between increased glutamatergic presynaptic bouton density and cognitive performance for both the Mini Mental State Examination (B, C) and Global Cognitive Test score (D, E). Data were grouped by cognitive ability as NCI, mAD, and sAD (B, D) or MCI, mAD, and sAD (C, E). Note that cognitive performance declines with decreasing glutamatergic presynaptic bouton density across all groupings and test types.

Figure 3.

Double-immunohistochemical staining of glutamatergic dystrophic neurites (brown) and amyloid plaques (blue) in the midfrontal gyrus brain region. Note the predominant localization of dystrophic neurites to the inner areas of amyloid plaques, as opposed to the plaque periphery. Note also the increase in abundance of glutamatergic dystrophic neurites with increasing plaque size (A–C). D–F show the manner in which the relative abundance (total area of occupation) of glutamatergic dystrophic neurites was determined. E, The transformed file, which, as previously described, increases the computer's accuracy of detection. F, The encircled quantified area, in which dark blue coloration shows elements that met the required inclusion and exclusion criteria (size, color, intensity, saturation, and roundness) and were therefore included in the quantification. The light blue coloration shows elements that failed to match the necessary criteria and that were therefore excluded. Note the close level of overlap between computer detection and dystrophic neurite abundance, as well as the omission of smaller terminals or plaque fragments. Scale bar, 10 μm.

Figure 4.

Graphic depiction of the association between glutamatergic dystrophic neurite abundance and amyloid plaque size in the midfrontal gyrus of subjects with MCI (A), mAD (B), sAD (C), or all three subject groups combined (MCI, mAD, and sAD; D). Note the positive association between increasing plaque size and increasing glutamatergic dystrophic neurite abundance in all three groups. Note also that amyloid plaque size appears to increase with disease progression, as does the strength of the Pearson's correlation, thus suggesting that increasing levels of fibrillar Aβ correlate with increased glutamatergic terminal toxicity.