The present study demonstrates for the first time that hippocampal infusions of the MARCKS peptide, consisting of amino acids 151 to 175, significantly impairs memory. This effect in rats tested in the 16-arm radial maze is dose-dependent with robust impairment at the dose of local hippocampal infusions of 10 μg/0.5μl/side (6.4 mM), and smaller, inconsistent impairments at lower doses. The impairment was also reversible: at least 48 h after the local infusion of the high dose of MARCKS151-175
, the animals' performance in the maze returned to normal. The MARCKS151-175
peptide, at the dose of 10 μg/site (6.4 mM), impaired both working and reference memories with larger working memory deficits. Although the dramatic impairment of working memory was accompanied by a significant increase in the response latency, the effect does not seem to be associated with the disturbances in the motor activity or food motivation. Our observations did not detect any sedative effect of the drug or slowness in behavior of the treated rats, nor did we observe in the rats reduced interest in the food reward. The animals moved quickly in the central maze arena and although they heavily marked the entered arms with saliva and urine, the rats hesitated at the arm entrances. Typically, animals mark the entered arm at the beginning of the training in the 16-arm radial maze. The markings help them to keep track of the entered and un-entered arms. Trained animals usually quickly collect food in the radial maze, not marking the arms, a behavior which relies on the spatial working memory. This observation implies that working memory impairment produced by MARCKS151-175
is rather a result of disruption of hippocampal mechanisms, which are critically involved in the spatial working memory regulation. In this regard, an in vitro
hippocampal investigation by Gay et al. (2008)
is of significant importance. The authors demonstrated that MARCKS151-175
acts as a powerful α7 nAChR antagonist and binds directly to the extracellular domain of nAChRs. They also found that a truncated form of the peptide, MARCKS159-165
, was unable to inhibit acetylcholine responses of the α7 nAChR. Consistent with this report, our current investigation found no effect of the truncated MARCKS peptide infused in the ventral hippocampus on the rat memory performance in the radial maze. Gay et al. (2008)
suggested that truncated, short forms of the MARCKS peptide could act in a competitive manner with the MARCKS ED peptide for binding to the nAChR, and thus could regulate nAChR signaling. They discovered that the MARCKS ED peptide binds to the α7 nAChR at a location distinct from the α-bungarotoxin binding site and acts as an allosteric antagonist rather than an open channel blocker. The authors proposed that MARCKS ED peptide could function as a reversible inhibitor under physiological conditions.
Nicotinic receptors are highly expressed in the brain with α7 nAChRs concentrated in the hippocampus (Kawai et al., 2002
; Lendvai and Vizi, 2008
; Placzek et al., 2009
; Tribollet et al., 2004
; Wada et al., 1989
), the structure critically involved in memory function. The critical role of α7 nAChRs in working memory has been also well documented in pharmacological studies with receptor antagonists (Bettany and Levin, 2001
; Hashimoto et al., 2008
; Levin et al., 2002
; Nott and Levin, 2006
; Pichat et al., 2007
; Thomsen et al., 2009
) as well as genetic studies with α7 nAChR knockout mice (Levin et al. 2009
). Our previous studies (Felix and Levin, 1997
; Levin et al., 2002
) demonstrated that acute infusions of α7 or α4β2 nicotinic receptor antagonists (methyllycaconitine, MLA or dihydro-β-erythroidine, DHβE, respectively) in the rat ventral hippocampus resulted in significant impairment of working and reference memory, with working memory affected more severely. The first investigation was done in the less challenging 8-arm radial maze. Such setting required higher doses of the nicotinic receptor antagonists (78.7 μg/side of MLA and 106.9 μg/side of DHβE) to affect memory performance. However, these doses also elicited convulsive manifestations. Therefore we repeated investigation in the more challenging 16-arm radial maze, which allowed to use lower sub-convulsant concentrations of the antagonists. We found that MLA at dose of 27 and DHβE at dose 6.75 μg/side caused a significant deficit in working and to a lesser extent in reference memory. To our surprise combined infusions of the two antagonists did not produce an additive effect with respect to memory impairment. Infusions of MARCKS151-175
at dose 10 μg/side produced even more powerful memory impairing effect in the similar settings (the effect was more consistent and higher in magnitude). Although MARCKS151-175
could antagonize both α7 and α4β2 nicotinic receptors, we suggest that MARCKS151-175
exerts its action through additional mechanisms different from inhibition of nicotinic receptors, given the previous work showing that combined α7 and α4β2 nicotinic receptor blockade did not cause additive effects.
MARCKS is a primary PKC substrate that binds plasma membrane via n-terminus myristoylation and electrostatic interaction with the phosphorylation site domain, binds calmodulin, binds and cross-links filamentous actin, all in PKC phosphorylation-reversible manner (Arbuzova et al., 2002
; Blackshear, 1993
). Based on these properties, MARCKS has been proposed to be an important intermediate transducer of receptor generated PKC activity into lasting alterations in membrane-actin cytoskeletal morphology (McNamara and Lenox, 2004
). In their recent work McNamara et al. (McNamara et al., 2005
) demonstrated that MARCKS overexpression was detrimental for cognitive function such as spatial learning. They suggested that elevation in the MARCKS:PKC ratio could produce a negative effect on specific aspects hippocampal function.
In neurons MARCKS is located to dendritic spines and to axonal terminals in association with synaptic vesicles (Ouimet et al., 1990
), and co-localizes with synaptophysin (Lu et al., 1998
). Notably, MARCKS binds calmodulin at low calcium concentrations and may sequester free calmodulin at the cell membrane (Verghese et al., 1994
). The principal role of calmodulin is to bind calcium. MARCKS, thus, could impair calcium-signaling processes associated with neurotransmitter release and memory function. In addition, an overexpression of nonphosphorylated MARCKS in Schaffer collateral terminals, which normally express MARCKS at low levels in the mature brain (McNamara and Lenox, 1997
) was suggested to enhance cross-linking of F-actin at the plasma membrane and thus diminish F-actin cytoskeletol plasticity necessary for maintenance of hippocampal long-term potentiation (McNamara et al., 2005
). Thus, memory impairing effect caused by acute infusions of MARCKS151-175
peptide in the rat ventral hippocampus could be a combined result of α7 nAChR inhibition and alteration of different cellular signaling mechanisms in the hippocampus.
The current study provides further evidence at behavioral level that effector domain MARCKS peptides could be involved in the regulation and impairment of cognitive functions. Future studies should explore the issue of a possible involvement of MARCKS peptides in human diseases of cognitive impairment.