Elsevier

Neurobiology of Disease

Volume 45, Issue 1, January 2012, Pages 381-387
Neurobiology of Disease

Preferential accumulation of amyloid-beta in presynaptic glutamatergic terminals (VGluT1 and VGluT2) in Alzheimer's disease cortex

https://doi.org/10.1016/j.nbd.2011.08.027Get rights and content

Abstract

Amyloid-beta (Aβ) is thought to play a central role in synaptic dysfunction (e.g. neurotransmitter release) and synapse loss. Glutamatergic dysfunction is involved in the pathology of Alzheimer's disease (AD) and perhaps plays a central role in age-related cognitive impairment. Yet, it is largely unknown whether Aβ accumulates in excitatory boutons. To assess the possibility that glutamatergic terminals are lost in AD patients, control and AD synaptosomes were immunolabeled for the most abundant vesicular glutamate transporters (VGluT1 and VGluT2) and quantified by flow cytometry and immunoblot methods. In post-mortem parietal cortex from aged control subjects, glutamatergic boutons are fairly abundant as approximately 40% were immunoreactive for VGluT1 (37%) and VGluT2 (39%). However, the levels of these specific markers of glutamatergic synapses were not significantly different among control and AD cases. To test the hypothesis that Aβ is associated with excitatory terminals, AD synaptosomes were double-labeled for Aβ and for VGluT1 and VGluT2, and analyzed by flow cytometry and confocal microscopy. Our study demonstrated that Aβ immunoreactivity (IR) was present in glutamatergic terminals of AD patients. Quantification of Aβ and VGluT1 in a large population of glutamatergic nerve terminals was performed by flow cytometry, showing that 42% of VGluT1 synaptosomes were immunoreactive for Aβ compared to 9% of VGluT1 synaptosomes lacking Aβ-IR. Percentage of VGluT2 synaptosomes immunoreactive for Aβ (21%) was significantly higher than VGluT2 synaptosomes lacking Aβ-IR (9%). Moreover, Aβ preferentially affects VGluT1 (42% positive) compared to VGluT2 terminals (21%). These data represent the first evidence of high levels of Aβ in excitatory boutons in AD cortex and support the hypothesis that Aβ may play a role in modulating glutamate transmission in AD terminals.

Highlights

► We quantify glutamatergic terminals in Alzheimer's disease and aged control brain. ► Glutamatergic boutons are fairly abundant in pathologic and aged control cortex. ► We examine levels of Abeta in glutamatergic boutons in Alzheimer's disease cortex. ► High levels of Abeta are found in excitatory boutons in Alzheimer's disease cortex.

Introduction

Alzheimer's disease (AD) is the most common form of dementia, affecting > 5 million Americans (Hebert et al., 2003). Abnormal amyloid-beta (Aβ) metabolism, tau hyperphosphorylation and synapse loss are characteristic features of AD. Although the molecular triggers of AD are unknown, extensive studies indicate that Aβ plays a principal role in AD pathogenesis (Kuo et al., 1996, McLean et al., 1999, Naslund et al., 2000).

Presynaptic buildup of soluble Aβ is suggested as a proximal cause of synapse dysfunction in AD (Fein et al., 2008, Shankar et al., 2007, Shankar et al., 2008). Natural human Aβ dimers are able to (i) mediate loss of dendritic spines and excitatory synapses; (ii) inhibit long-term potentiation (LTP) and facilitate long-term depression (LTD) in normal rodent hippocampus, and (iii) impede learned behavior in normal rats (Shankar et al., 2007, Shankar et al., 2008). The degree of clinical impairment strongly correlates to the decrease of electrophysiologically active synapses especially glutamatergic pyramidal synapses (Shankar et al., 2007, Shankar et al., 2008, Terry et al., 1991). Nonetheless, a direct link between presynaptic Aβ and the loss of excitatory function has yet to be established.

Glutamate is a key neurotransmitter in primary perception and cognition. It is the principal excitatory neurotransmitter in the neocortex and hippocampus, major brain areas affected in AD. Furthermore, glutamate is associated with excitotoxicity and LTP. Despite the central role of glutamatergic synapses in learning and memory, their involvement in AD pathology remains unclear. Many neurochemical studies have associated a dysfunction of the glutamatergic system with AD pathology, including reduction of glutamic acid content in AD brain (Procter et al., 1988), reduced receptor binding (Cross et al., 1987) and decreased cortical and hippocampal glutamate uptake which was interpreted as a result of glutamatergic synapse loss (Cowburn et al., 1988, Hardy et al., 1987). Recently, synthetic Aβ peptide species were shown to potentiate K+-induced glutamate release from normal rodent hippocampus (Kabogo et al., 2010).

With the availability of VGluT specific antibodies, immunohistochemical investigation of excitatory boutons has become possible. VGluT1 and VGluT2 are presynaptic proteins responsible for glutamate transport into synaptic vesicles and are found in functionally different subtypes of neurons (Fremeau et al., 2004, Herzog et al., 2001, Takamori et al., 2000, Varoqui et al., 2002). Several immunohistochemical studies yielded conflicting results in VGluT1 and VGluT2 expression in AD post-mortem brain tissue. Glutamatergic presynaptic bouton density has been reported as elevated in midfrontal gyrus of patients with mild cognitive impairment (MCI) and depleted in mild- and severe stage of AD (Bell et al., 2007). Another group observed an important reduction in VGluT1 and VGluT2 expression in AD prefrontal cortex; VGluT1 reduction was correlated to the clinical dementia rating score (Kashani et al., 2008). Others found reduced VGluT1 levels in parietal and occipital cortex but not in frontal cortex of AD patients; VGluT2 expression was not modified in these brain areas (Kirvell et al., 2006). In a more recent investigation, VGluT1 expression and cognitive scores were correlated but no difference was observed in VGluT1 expression in prefrontal and temporal cortex of control and AD patients (Kirvell et al., 2010). Because of the discrepancy between studies, the involvement of excitatory synapses in AD remains unclear, despite their central role in clinical symptoms that characterize the disease.

In this study, we aimed to (i) detect alterations in glutamate transporter levels by examining large population of AD and control synaptosomes using flow cytometry analysis, and (ii) determine the degree to which Aβ is present in excitatory terminals. In the present experiments, Aβ, VGluT1 and VGluT2 were quantified by flow cytometry and confocal microscopy in surviving terminals of AD parietal cortex (Brodmann area A7). This area of the CNS was previously reported to be severely affected in AD and abundantly expressing Aβ (Fein et al., 2008, Kirvell et al., 2006).

Section snippets

Human brain specimens

A total of 15 individuals were studied including 8 with AD (7 females, 1 males, age 86.0 ± 3.3 year; mean postmortem delay: 6.9 ± 0.8 h) and 7 aged cognitively normal controls (4 females, 3 males, age 89.4 ± 3.3 year, mean postmortem delay: 7.4 ± 1.4 h). Brain specimens from parietal cortex (Brodmann area A7) were obtained at autopsy from the Alzheimer's disease Research Centers at the University of Southern California, the University of California at Los Angeles and the University of California at Irvine.

Quantification of VGluT1 and VGluT2 terminals in human parietal cortices

As synaptic terminals are not well resolved by light microscopy, even at the highest magnification, we analyzed immunolabeled synaptosomes by flow cytometry (FACS). This method quantifies multiple parameters on each nerve terminal in a sample (Fein et al., 2008), including fluorescence and forward scatter (FSC), which is proportional to the size of a particle. We have previously reported that non-synaptosomal elements are excluded by drawing a size-based gate that includes only particles that

Discussion

Glutamate dysfunction mediated by Aβ oligomeric species is thought to be an important aspect of AD pathogenesis but the underlying mechanisms remain intangible. Abnormal synaptic plasticity induced by Aβ is also attributed to early memory loss that precedes neuronal degeneration (Selkoe, 2002). Several studies reported the co-localization of Aβ with postsynaptic NMDA-receptors (Dewachter et al., 2009, Lacor et al., 2007) but it remains unclear whether Aβ is present within presynaptic

Conclusion

Our report demonstrates the presence of Aβ in cortical glutamatergic terminals and underlies their important contribution in AD pathology. These findings emphasize the importance of elucidating the synaptic cascade triggered by Aβ in order to prevent glutamate dysfunction and cognitive loss. This study supports the valuable therapeutic benefits of targeting glutamatergic neurons to treat Alzheimer's disease.

Acknowledgments

We are thankful Mr. David Moom for technical assistance. This work was supported by NIH AG27465 to KHG, by NIA AG18879 to CAM. HVV is supported by the Daljit S. and Elaine Sarkaria Chair in Diagnostic Medicine. SS is supported by UCLA School of Nursing intramural grants and a pilot grant from NIA/Mary S. Easton Center for Alzheimer's Research at UCLA (P50 AG016570). Tissue was obtained from the Alzheimer's Disease Research Center Neuropathology Cores of USC (NIA 050 AG05142), UCLA (NIA P50 AG

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