Stereologic estimates of total spinophilin-immunoreactive spine number in area 9 and the CA1 field: Relationship with the progression of Alzheimer's disease
Introduction
Alzheimer's disease (AD) is a progressive, degenerative disorder of the central nervous system. The major hallmarks of AD include the presence of extracellular amyloid deposits and intracellular neurofibrillary tangles (NFT) (Alzheimer, 1907, Braak and Braak, 1991, Dickson et al., 1988, Terry et al., 1964, Yamaguchi et al., 1988). Several studies have shown that in addition to these traditionally described lesions, AD is characterized by selective neuronal loss (Hof et al., 1990, Terry et al., 1981), severe and early loss of synapses (Davies et al., 1987, DeKosky and Scheff, 1990, Hamos et al., 1989, Masliah et al., 1989, Scheff et al., 2006) and synaptic pathology (Masliah et al., 1991a, Masliah et al., 1991b). Early immunocytochemical studies indicated an average 45% decrease in presynaptic terminal density in the AD neocortex (Masliah et al., 1989, Masliah et al., 1991b, Weiler et al., 1990). Quantitative morphometric study of temporal and frontal cortical biopsies performed within an average of 2–4 years from the onset of clinical AD revealed 25–35% decrease in the numerical density of synapses and 15–35% decrease in the number of synapses per cortical neuron (Davies et al., 1987). In terms of clinicopathologic correlations, much of the previous work has focused on loss of presynaptic markers such as synaptophysin (Dickson et al., 1995, Masliah et al., 2001, Masliah and Terry, 1993, Scheff and Price, 2003, Sze et al., 1997, Terry et al., 1991). The contribution of Terry et al. (1991) first implied that severity of AD is more robustly related to synapse loss than amyloid plaques, NFT densities, degree of neuronal loss or extent of cortical gliosis. In particular, they postulated that synaptophysin decrease in the prefrontal cortex is the major correlate of cognitive deficits, explaining about 70% of the global psychometric test variability (Terry et al., 1991). Recent reports revealed synaptophysin immunoreactivity reduction in NFT-containing neurons in the hippocampus and association cortices in minimal cognitive impairment and early AD, pointing to the relationship between NFT formation and loss of presynaptic markers (Callahan and Coleman, 1995, Heinonen et al., 1995, Masliah et al., 2001, Sze et al., 1997). However, estimates of synaptic loss in these studies relied upon density measures and were based on two unwarranted assumptions, namely that the size of the region under analysis remains constant across diagnostic groups and that synaptic size does not change (Chételat and Baron, 2003, Scheff and Price, 2003, Wolf et al., 2003).
In contrast to presynaptic markers, AD changes in postsynaptic structures have been less studied. Although postsynaptic components are thought to be affected in early-onset AD (Davidsson and Blennow, 1998, Spires et al., 2005), few studies have explored in humans the status of dendritic spines in brain aging. Spines are dynamic structures that are the proposed site of synaptic plasticity underlying learning and memory (Horn et al., 1985, Moser et al., 1994, Segal, 2002). Because of the distance of dendritic extent from the soma, dendritic spines may be particularly vulnerable to incipient degenerative processes that disrupt intracellular signaling and synaptic functions. It has been hypothesized that alterations in synaptic activity can cause morphologic changes in dendritic spines (Harris, 1999, Maletic-Savatic et al., 1999, McKinney et al., 1999). Conversely, morphologic changes in dendritic spines (Nimchinsky et al., 2002, Shepherd, 1996, Yuste and Denk, 1995) have profound effects on the electrical and biochemical properties of synapses (Denk et al., 1996, Svoboda et al., 1996) and may regulate the efficacy of synaptic transmission (Yuste and Denk, 1995).
Spinophilin, also called neurabin II (Satoh et al., 1998), is a synaptic protein implicated in spine formation and synaptic transmission in different types of dendritic spines and at excitatory and some inhibitory postsynaptic sites on dendritic shafts (Muly et al., 2004, Ouimet et al., 2004). Spinophilin displays a remarkably distinct localization to the heads of majority of dendritic spines in all brain regions examined, although the concentration per spine is regionally and locally variable. Spinophilin immunoreactivity has been shown to be intense in the majority of dendritic spines of rat hippocampus (Allen et al., 1997). It is present in about 93% of the dendritic spines in rhesus monkey hippocampus (Hao et al., 2003), but sparsely distributed in other portions of the dendrites, making it an excellent marker for quantitative assessment of spine numbers (Hao et al., 2003). In order to explore the role of dendritic spine loss in cognitive decline, we performed a stereological analysis of spinophilin-immunoreactive puncta in the CA1 and CA3 fields of the hippocampus and area 9 in 18 elderly individuals prospectively assessed with the Mini Mental State Examination (MMSE) and Clinical Dementia Rating (CDR) scale.
Section snippets
Patients
The brains from 18 elderly patients, presenting with either normal aging or with various degrees of cognitive impairment were obtained at autopsy within 30 h of death. Clinical data were obtained from the medical records of the patients and the neuropathologic evaluation from the Departments of Geriatrics and Psychiatry, University of Geneva School of Medicine, Geneva, Switzerland and the Department of Psychiatry, Mount Sinai School of Medicine, New York, USA (Table 1). All cases underwent
Distribution of spinophilin immunoreactivity
Qualitatively, intense spinophilin immunoreactivity was observed in the hippocampus and in all six cortical layers of area 9 as a pattern of bright puncta (∼0.5–1 μm in diameter) in both controls and AD patients. Within the CA1 field, the immunostaining was most intense in the stratum oriens and stratum radiatum, weaker in the stratum lacunosum-moleculare, and faint around cell bodies in the stratum pyramidale (Fig. 1A). The pattern of immunoreactivity in the hippocampal formation provided clear
Discussion
The present study reports unbiased stereologic assessment of total spinophilin-immunoreactive profiles in AD cerebral cortex and used multivariate regression models to estimate the contribution of dendritic spine loss in cognitive decline after controlling for the severity of NFT formation. Because synaptic loss may be related to the NFT development in both local and distally projecting neurons, we used the Braak NFT staging that makes it possible to assess the whole burden of NFT pathology in
Conflict of interest
None of the authors declare an actual or potential conflict of interest.
Acknowledgements
We thank Drs. Patrick Allen and Paul Greengard for generously providing the anti-spinophilin antibody, and Bridget Wicinski, Ginelle Andrews and William G.M. Janssen for expert technical assistance. This work was supported by grants AG02219 and AG05138 from the National Institutes of Health, Bethesda, MD, USA (PRH, JHM, DPP, VH) and the Jérôme Tissières Foundation, Geneva, Switzerland (PG).
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