Age-related cognitive decline affects a significant portion of the healthy aging population but its causes remain to be determined. Since neuronal communication underlies cognitive function, faulty synaptic communication leading to impaired neurotransmission is likely a key factor in age-related cognitive deficits. In the aged hippocampus, neuronal activation during behavioral tests of spatial learning and memory capacities is significantly depressed, and electrophysiological correlates of learning and memory are dysregulated (Norris et al. 1996
;Norris et al. 1998
;Rosenzweig and Barnes 2003
). Age-related alterations in hippocampal transcript and protein content that may contribute to impaired synaptic function have been identified for synaptic signaling proteins (Baxter et al. 1999
;Jiang et al. 2008
;Liu et al. 2008
;Majdi et al. 2009
;Sato et al. 2005
;Shi et al. 2007
Hippocampal synaptosomes isolated from Wistar rats demonstrated significant decreases in SNAP25 and synaptophysin expression between adult (12 months) and aged (18–24 months) animals, with magnitudes of change similar to those observed here (Canas et al. 2009
). Likewise, synaptic vesicle glycoprotein 2B, SV2-related protein, Homer 1, and synaptoporin were recently demonstrated to decrease in expression throughout aging in Fischer 344 rats of ages similar to those in this study (i.e., 3 months, 12 months, 23 months) (Kadish et al. 2009
). These findings are in agreement with reports of dysregulated synaptic connectivity and functionality with aging (Kumar et al. 2007
;Sametsky et al. 2008
;Thibault et al. 2001
). Many of these studies, however, have focused on specific transcripts and protein species rather than on the entire proteomic profile of hippocampal synapses. The work presented here adds to the understanding of age-related alterations in the unfractionated hippocampus by focusing on the proteomic composition of the hippocampal synaptoproteome.
In this study, age-related downregulation of 14-3-3 signaling protein isoforms and a number of proteins with neurotransmission-regulating functions were identified by bioinformatic analysis to be members of a protein network implicated in nervous system function. Alterations in synaptic protein expression have the potential to decrease stimulus-induced neurotransmission and replenishment of synaptic vesicle pools during instances of prolonged or intense stimulation, such as those necessary for learning and the formation and maintenance of memory ().
Functional roles of neurotransmission-regulating proteins altered with increasing age
A salient finding of this work was the biphasic expression pattern of hippocalcin, a calcium-binding protein required for spatial learning and memory (Kobayashi et al. 2005
). Hippocalcin facilitates calcium-mediated LTD in ex vivo
hippocampal slices, in which calcium signaling and calcium channel expression are also dysregulated (Brewer et al. 2007
;Landfield and Pitler 1984
). Additionally, hippocalcin functions as a diffusible calcium sensor critical in calcium gating of slow afterhyperpolarization in hippocampal neurons (Tzingounis et al. 2007
). Although specific mechanisms underlying the involvement of hippocalcin in synaptic plasticity are not fully understood, depolarization-sensitive calcium-induced translocation along hippocampal dendrites and axons and interactions with clathrin-mediated endocytic machinery and glutamate receptors are likely contributing factors (Markova et al. 2008
;Palmer et al. 2005
). Interestingly, we observed a significant decrease in synaptosomal hippocalcin expression in Adult rats compared to Young-adult rats, at ages prior to the development of hippocampal cognitive deficits. In contrast, in Aged rats, hippocalcin returned to levels observed in Young-adults. It is possible that, in conjunction with abnormal calcium dynamics and decreased neurotransmission-regulating proteins, increased synaptic expression of hippocalcin facilitates the susceptibility of aged rodents to LTD and impaired spatial learning and memory.
The 14-3-3 isoform family of scaffolding adaptor proteins is highly enriched in brain and is implicated in modulation of neurotransmission, with potential roles in learning and memory (Broadie et al. 1997
;Li et al. 2006
;Philip et al. 2001
). Through association with hundreds of binding partners, 14-3-3 isoforms mediate numerous processes including phosphatase signaling, protein trafficking and conformational changes, and facilitation of signal transduction of exocytic pathways (Pozuelo et al. 2004
). In hippocampal neurons, 14-3-3 activity reduces short term synaptic depression by modulating calcium channel inactivation dynamics (Li et al. 2006
). Recent proteomic analyses of 14-3-3 binding proteins suggest a postsynaptic component of 14-3-3 function in neurotransmission. PSD95, a postsynaptic scaffolding protein highly expressed in hippocampal glutamatergic synapses, binds 14-3-3 and enables indirect interaction of 14-3-3 with numerous glutamate receptors and synaptic signaling proteins (Fernandez et al. 2009
). Similarly, 14-3-3 modifies postsynaptic glutamate receptor signaling through interaction with Homer 3, a postsynaptic scaffolding protein that links receptor signaling targets and receptors (Angrand et al. 2006
). Decreased synaptosomal expression of multiple 14-3-3 isoforms was identified by proteomic and immunoblotting techniques in this study, indicating a potential mechanism for disrupted protein-protein interactions required for maintenance of healthy neurotransmission.
A large subset of protein expression changes in this study, such as NSF, SNAP25, syntaxin 1, VAMP2, synaptophysin, synapsin 1, dynamin 1, amphiphysin, clathrin, syndapin 1, and syntaxin-binding protein 1, represent both effectors and regulators of neurotransmission. For example, SNAP25, syntaxin 1, and VAMP2 interact to form the SNARE complex critical for vesicle docking and fusion. Heterozygous loss of SNAP25 decreases stimulus-evoked membrane fusion and impairs short term plasticity and spatial learning at both excitatory and inhibitory neurons (Tafoya et al. 2006
;Washbourne et al. 2002
). Similarly, disrupted expression of VAMP2, the vesicular SNARE component, nearly abolishes calcium-induced exocytosis and endocytic replenishment of the synaptic vesicle pool (Deak et al. 2004
;Schoch et al. 2001
). Synapsin 1, also decreased with age, crosslinks neurotransmitter-primed synaptic vesicles to the cytoskeleton in the resting state to effectively restrain the reserve pool and minimize spontaneous vesicle mobilization in a phospho-dependent manner. Synapsin 1-knockout mice exhibit demonstrate impaired cognitive function and spatial memory with age (Corradi et al. 2008
). Synaptophysin and PSD95 which undergo age-related changes in expression in both humans and animal models (Adams et al. 2008
;Head et al. 2009
;Majdi et al. 2009
), were also reduced in synaptosomal expression with age in this work. Together, the synaptoproteomic changes observed in this study suggest an age-related impairment of exocytosis (SNARE proteins, NSF, synapsin 1, tropomodulin 2), endocytosis (clathrin, dynamin, amphiphysin, syndapin, etc.), and receptor aggregation (PSD95). Additionally, several of these proteins are implicated in activity-dependent synaptic maintenance.
This study describes a coordinated downregulation of a network of synaptosomal proteins with functions in neurotransmitter vesicle exocytosis and recycling dynamics. These proteins are often absent from proteomic reports of aging, perhaps due to their relatively low whole-tissue expression levels. The combination of synaptosome enrichment and 2-DIGE proteomic methods represents a technical advance that enabled the assessment of the protein composition of an isolated subcellular region, the synaptic terminal, allowing quantitation of neurotransmission-related proteins in their functional niche. Synaptosomal enrichment also increased sensitivity for less-abundant protein species that often fall below the limit of detection in proteomic studies of unfractionated tissue. Additional novel pathways and networks revealed by this synaptoproteomic analysis remain to be pursued in future studies. Also, in agreement with previous reports, we identified a number of metabolic and mitochondrial proteins differentially regulated in comparisons of Young-adult, Adult, and Aged rats (Freeman et al. 2009
;Poon et al. 2006b
). The synaptosomal preparation used in this study, although highly enriched for synaptic terminals, also contains both synaptic and non-synaptic membrane fragments and mitochondria. Definitive localization studies are required to determine whether the changes in expression of the specific mitochondrial proteins occur throughout the cell or are restricted to specific subcellular compartment.
Together, observed decreases in effectors and regulators of neurotransmission, including SNARE and SNARE-associated proteins and 14-3-3 isoforms, suggest an age-based deterioration of hippocampal neurotransmission that occurs between adulthood and advanced age. These alterations in synaptic protein expression have the potential to decrease stimulus-induced neurotransmission and replenishment of synaptic vesicle pools during instances of prolonged or intense stimulation, such as those necessary for learning and the formation and perseverance of memory.