Chronic neuroinflammation is consistently present during the early stages of Alzheimer's disease and HIV-associated dementia and likely contributes to the cognitive dysfunction associated with these disorders and drives disease progression [1
]. It has hence become important to understand the mechanisms underlying chronic neuroinflammation-induced cognitive impairments and to explore potential therapeutic approaches to ameliorate them. Chronic neuroinflammation can be reliably reproduced in rodents by the slow, continuous infusion of a low dose of LPS into the brain, and such models are associated with impaired spatial cognitive function [11
]. Herein we were able to demonstrate that the blood-brain barrier permeant TNF-α lowering agent, DT, effectively rescued hippocampus-dependent cognitive impairment induced by chronic neuroinflammation. The decrease in TNF-α mRNA was paralleled with a normalization of the inflammation-induced disproportionate expression of the plasticity-related immediate-early-gene Arc. These data suggest that the proinflammatory cytokine TNF-α signaling is critically involved in the disruption of patterns of hippocampal activity underlying learning and memory and that inhibition of TNF-α synthesis can restore cognitive function from a behavioral and cellular prospective.
Chronic neuroinflammation is characterized by a long-standing activation of immune cells and subsequent sustained release of proinflammatory factors. Here chronic neuroinflammation was induced by slow intraventricular infusion of LPS, a component of the outer membrane of Gram-negative bacteria, known to selectively activate microglial cells through the stimulation of TLR4/CD14 receptors [4
]. This treatment gave rise to a long-lasting innate immune response that persisted in the hippocampus even following discontinuation of LPS infusion, as evidenced by increased gene expression of the inflammatory markers TNF-α, IL-1β, TLR2 and TLR4. DT was administered after discontinuation of LPS infusion to define the ability of the compound to reverse neuronal dysfunction and cognitive impairments induced by self-sustained chronic neuroinflammation.
Moreira and co-workers described that thalidomide reduced the half-life of TNF-α mRNA by approximately 50% [32
]. Latterly, Zhu and Greig and co-workers illustrated that thiol analogs of thalidomide, including DT, were more potent than thalidomide at lowering LPS stimulated TNF-α protein secretion in vitro
. Additionally, the thiol analogs were found to share the same mechanism of action to that of thalidomide where the agents acted upon the 3'-UTR of TNF-α mRNA [35
]. Interestingly, several protein mRNA's are subject to regulation at the 3'-UTR region via actions upon adenylate/uridylate (AU)-rich elements. TNF-α mRNA is known to be regulated by several RNA binding proteins such as HuR which stabilizes RNA and effectively increase the translation efficiency of the cytokine [48
], tristetraprolin and AUF1 both of which destabilize RNA and thus reduce the translation of RNA into protein [49
]. Both HuR and tristetraprolin are regulated by p38 mitogen-activated protein kinase (p38 MAPK) and p38 MAPK has been shown to be inhibited by thalidomide [51
]. It is likely that DT acts in a similar manner, however additional studies will be required to establish this.
The chosen dose and duration of DT administration were based on preliminary results showing that 14 days of DT administration (56 mg/kg/day; i.p.) reduced microglia activation and prevented cognitive impairments induced by concomitant LPS infusion (Tweedie D, Rosi S, Greig NH, unpublished work). The dose of DT (56 mg/kg, equimolar to 50 mg/kg of thalidomide) compares favorably with those of thalidomide used in humans, where doses of up to 1200 mg are administered. Hippocampal gene expression analysis showed a normalization of TNF-α levels in LPS-infused rats treated with DT and confirmed the compound's ability to access the CNS and to act as a TNF-α synthesis inhibitor, in line with previous studies [34
]. The increase in TNF-α levels and its normalization by DT were paralleled by similar modulation of TNFR2. TNFR2 is known to be expressed primarily by cells of the immune system (including microglia) and by endothelial cells and to act via a number of different signaling pathways to increase NFκB-mediated transcription of anti-apoptotic and pro-inflammatory gene targets [17
]. The normalization of TNF-α by DT was not associated with major variations of other cytokines and chemokines known to regulate inflammation (IL-1β, IL-6, IL-4, CD200 and CX3CL1). Importantly, IL-1β levels remained significantly elevated, suggesting that DT effects were mediated primarily through TNF-α but not IL-1β mediated signaling pathway inhibition.
TNF-α has been shown to promote the proliferation and activation of microglia cells [17
]. Therefore, we investigated whether microglia activation could be modified by DT treatment, after the initiation and onset of microglial activation. We observed that DT treatment did not impact the number of MHC-Class II positive microglia accumulated within the DG and CA3 regions of the hippocampus in response to prior LPS-infusion. The elevated expression of genes involved in the TLR-mediated signaling pathways has been reported to be indicative of classical microglia activation following a central immune challenge [41
]; in line with these findings, our data showed increased expression of TLR2 and TLR4 levels in LPS-vehicle rats. Importantly, normalization of TNF-α transcripts by DT was paralleled by a significant decline in the expression of several genes involved within the TLR mediated signaling pathways, such as TLR2, TLR4, Hmgb1 and IRAK1. As it is established that TLR-mediated signaling pathway activation leads to increased TNF-α expression [54
], we can hypothesize that this, in turn, contributed to the decrease in TNF-α expression observed in our experiment. As MHC class II expression can also occur in microglia that are alternatively activated to produce anti-inflammatory cytokines, we also studied the expression of the anti-inflammatory cytokine IL-4; however no differences were observed across treatment groups. Our findings suggest that the decrease of TNF-α led to an attenuated expression of genes involved within the TLR-mediated signaling pathway associated with classical microglia activation.
Rats with chronic neuroinflammation displayed impaired performances in both novel place recognition and spatial learning and memory retention tests, but not in the novel object recognition test. The processing of spatial recognition memory and the ability to remember where an event occurs within an allocentric place have been shown to be highly dependent on the hippocampus [43
]. In contrast, findings from several lesion and imaging studies suggest that the ability to recognize an object that was part of a previous recent event is not hippocampus dependent but relies on the perirhinal cortex [43
]. In this context, our data therefore indicate that chronic neuroinflammation impaired hippocampus-dependent but not hippocampus-independent cognition. This behavioral phenotype is highly consistent with the fact that neuroinflammation is primarily distributed within the hippocampus in our model [11
] and further demonstrates the high vulnerability of the hippocampal formation toward neuroinflammation, as is also the case in humans. Importantly, the cognitive deficits observed in this study were observed up to 3 weeks after LPS infusion ended, which is in accord with our recent study showing that neuroinflammation persists for at least 2 months following termination of LPS-infusion [13
]. Taken together, our data suggests that, once initiated, neuroinflammation time-dependently persists, significantly impacts neuronal function and leads to hippocampus-dependent cognitive deficits.
Importantly, chronic neuroinflammation resulted in both short- and long-term spatial recognition memory deficits as measured following 5-min and 24-h delays. Short-term memory relies on synaptic mechanisms that do not require protein synthesis and is insensitive to inhibitors of transcription and translation. In contrast, storage of long-term memory, or memory consolidation, requires protein synthesis-dependent changes in synaptic strength [59
] and the expression of the plasticity related immediate early gene Arc [64
]. Thus, our data support the concept that both protein synthesis-independent and protein synthesis-dependent forms of synaptic plasticity were altered by chronic neuroinflammation. In addition to its well-known role in inflammation [17
], TNF-α has direct effects on glutamate transmission and, notably, has been shown to increase AMPA receptor surface expression [20
]. The trafficking of AMPA receptors is thought to underlie, at least in part, both the rapid form of synaptic plasticity and long-lasting and protein synthesis-dependent changes in synaptic strength [67
]. In line with previous reports [68
], our findings suggest that prolonged induction of TNF-α during chronic neuroinflammation may contribute to a dysregulation of synaptic homeostasis causing short-term recognition and long-term spatial memory deficits.
Inhibition of TNF-α synthesis by DT was able to restore the 5 min delay spatial recognition memory, indicating that normalization of TNF-α levels was associated with a significant amelioration of synaptic homeostasis underlying short-term memory. In contrast, normalization of TNF-α was not sufficient to restore the 24 h delay spatial recognition. The difference in the ability of DT to rescue the 5 min but not 24 h delay recognition memory likely was not due to variations in its bioavailability, because the DT intraperitoneal injections were performed daily at the same time during the novel place recognition protocol and all tests were conducted at similar hours every day. Interestingly, inhibition of TNF-α by DT was able to restore both spatial learning and 24 h retention probe trial performance in the Morris water maze. The apparent discrepancy between the rescue by DT of long-term spatial memory in the Morris water maze but not in the novel place recognition may be due to differences between these tasks, such as the nature of the motivation or the amount of training performed. Indeed, although both assess long-term spatial memory, the Morris water maze performance relies on escape behavior, speed and accuracy of spatial navigation whereas novel place recognition relies on a novelty spontaneous preference paradigm. The amount of training also significantly differs between these tasks and was more important for the Morris water maze than for novel place recognition (see methods). We have recently demonstrated that additional training in the water maze paradigm can, in fact, mitigate initial mild cognitive deficits [70
]. Finally, the Morris water maze task was conducted subsequent to the novel place recognition test and rats possibly gained ability to process spatial information through their behavioral training. Thus, any of these variables could potentially interact with the consequences of DT treatment in ways that a beneficial effect was evidenced in the 24 h delay probe trial of the Morris water maze probe trial but not in the 24 h delay novel place recognition.
The plasticity-related immediate-early gene Arc
and its protein product are induced in hippocampal neurons following spatial exploration in percentages similar to those recorded electrophysiologically [12
is involved in the trafficking of AMPA glutamate receptors [73
] and Arc protein is required in the engagement of durable plasticity processes that underlie memory consolidation [64
]. In the present study, we used the detection of Arc as a reliable method for monitoring cellular activity reflecting spatial and contextual information processing in response to behavior (i.e. the Morris water maze probe trial) [12
]. We previously demonstrated that chronic neuroinflammation disrupts the expression pattern of behaviorally-induced Arc in the DG and CA3 after novel environment exploration [11
]. Both the DG and CA3 hippocampal subregions cooperate to efficiently process spatial information [74
]. The CA3 area is thought to be particularly involved in large scale spatial representation, and lesions of the CA3 causes extensive spatial processing difficulties [74
]. Here, we report that the percentage of pyramidal neurons expressing Arc in the CA3 area was significantly dysregulated in LPS-vehicle rats with impaired memory performance. Importantly, the beneficial effect of DT on cognitive function was paralleled by a normalization of Arc de novo
protein synthesis expression. As Arc transcription is regulated by AMPA receptors [76
], we can hypothesize that the prolonged TNF-α increase contributes to the disruption in expression of behaviorally-induced Arc through a dysregulation of glutamate signaling homeostasis. It is thus conceivable that the reduction of TNF-α levels by DT did restore ideal Arc expression levels necessary to maintain optimal synaptic plasticity. The close correlation between Arc expression levels and probe trial performance across all experimental groups further strengthens the significance of Arc as a reliable molecular marker of neuronal plasticity related to memory.