3.1. Insights from Animal Behavior Investigations
The idea that 5-HT
4Rs agonists are promising drug candidates for memory impairments—especially those related to hippocampal dysfunction—was firstly supported by behavioral studies on different animal models
[42][43]. On one hand, cognitive impairments were often reported following antagonism (either pharmacologic agent or optogenetic construct) of 5-HT
4Rs
[44]. Surprisingly, the genetic ablation of 5-HT
4Rs did not alter learning and memory capacities in mice. However, the deleterious effect of scopolamine (a cholinergic antagonist) on long term memory was enhanced in 5-HT
4Rs KO mice
[45]. On the other hand, a very large number of preclinical studies reported consensual data supporting the beneficial effects of 5-HT
4Rs activation on memory performance. Overall, administration of 5-HT
4Rs agonists increased the learning rate in a hippocampus-dependent spatial task, such as the MWM
[46] and the object recognition test
[47][48][49]. 5-HT
4Rs agonists also restored memory impairments in animals treated with cholinergic antagonists
[50][51][52], in aged animal
[47][53] and in transgenic models of neurological diseases
[54][55]. Additionally, it was recently reported that intra-hippocampal injection of a 5-HT
4Rs agonist reduced sleep deprivation-induced memory impairments
[56]. These behavioral effects of 5-HT
4Rs modulation were extensively reviewed
[43][57]. Likewise, chronic 5-HT
4Rs activation was found to counterbalance learning and memory deficits induced by stress-induced depression
[3].
Additionally, 5-HT
4Rs have also been considered as an associative target of choice. Indeed, given the multidimensional and complex aspect of the pathogenesis of memory disorders, a new approach has emerged that consists of the simultaneous modulation of more than one target. After having proved the efficacy of 5-HT
4Rs stimulating activity in co-administration protocols with different AchE inhibitors
[48][58], the first multi-target drug ligand (MTDL) associating both activities has been designed. Named as Donecopride, this drug candidate was mainly developed for application in the field of AD
[59]. Indeed, these promising results argue for the development of other MTDLs combining 5-HT
4Rs agonistic activity with a different secondary target (other than AchE inhibitor) to be used for different medical application
[60].
These observations constitute the first line of evidence for an interest in 5-HT
4Rs activation in disorders related to hippocampal dysfunction. However, a limitation of preclinical research has certainly been the lack of investigation of 5-HT
4Rs’ functional and/or expression alteration in animal models that display memory deficits
[61]. In order to clarify if 5-HT
4Rs changes are causative or involved in the etiology of diseases, their expression pattern needs to be assessed on a cellular level in preclinical models.
3.2. Distribution of 5-HT4Rs in CNS and Memory Disorders
The distribution of 5-HT
4Rs within the brain is mainly restricted to the limbic system, thus intimately tied to memory function. The highest 5-HT
4Rs mRNA levels and densities are found in caudate, putamen, accumbens, and in the hippocampal formation
[62][63][64]. Within the hippocampal formation, the highest expression is found in the granule cell layer of the DG, followed by the pyramidal cell layer of the CA. Further, 5-HT
4Rs exhibit a layered distribution within CA subfields, with the highest densities identified in the stratum oriens and stratum radiatum. This suggests a localization of receptors at both basal and apical dendritic fields of pyramidal cells. Radio-ligand assays also show strong labelling in the stratum lucidum of the CA3 area, probably reflecting the presence of 5-HT
4Rs on MF
[65][66].
Ligand binding studies also help to reinforce the idea that 5-HT
4Rs play a pivotal role in memory function. In fact, the hippocampal density of 5-HT
4Rs was found to be inversely correlated with episodic memory test performance in healthy subjects
[67]. Further, it has also been observed that a striking feature of aging is the dramatic decrease in 5-HT
4Rs density that occurs
[63][68]. Likewise, the loss of 5-HT
4Rs expression was also observed in different cohorts of patients suffering from memory deficits
[61][69] and was correlated with the stage of the disease. For instance, a post-mortem brain analysis in AD patients reported a 70% decrease in hippocampal 5-HT
4Rs
[70], a change that was positively correlated to amyloid beta peptide load
[68]. Additionally, reduced 5-HT
4Rs binding was observed in the hippocampus in an animal model of depression
[71] (
Figure 2).
Moreover, it has been proposed that improvement of memory performance in patients who suffer from memory disorders is supported by up-regulation of 5-HT
4Rs, which in turns stimulates hippocampal 5-HT release as shown in rodents
[72][73] (
Figure 2). Indeed, there is now a large body of preclinical data showing a dynamic positive correlation between central 5-HT levels and 5-HT
4Rs densities. For instance, 5-HT
4Rs KO mice have diminished tissue levels of 5-HT (and its main metabolite, 5-HIAA)
[74]. Hence, 5-HT
4Rs activation could enhance 5-HT global tone through the positive feedback loop projecting from the prefrontal cortex to the DRN and thus, to the hippocampus
[61]. If so, this could account for the variation of 5-HT
4Rs expression observed in AD. Indeed, an upregulation of 5-HT
4Rs expression occurs at the pre-clinical stage of the disease and continues along with dementia progressing (up to mild stage), as if a compensatory strategy was put in place (in response to decrease in interstitial 5-HT levels), until exhaustion
[68]. Indeed, the loss of serotonergic cells in AD patients can reach above 70% in the DRN and MRN
[75] and can even be reduced to undetectable levels
[76][77]. This ultimately contributes to a decrease in hippocampal 5-HT neurotransmission, which has been identified as a correlate of cognitive impairment
[78] (
Figure 2). Altogether, the changes in 5-HT
4Rs density may reflect the abnormal range of 5HT levels required for memory functioning. Hence, the clinical stage of the disease during which 5-HT
4Rs may be used appears critical.
3.3. Morphological/Structural Alterations of Hippocampal Formation in Memory Disorders
Although a host of brain changes are likely to be responsible for cognitive decline, structural and functional hippocampal alterations were identified as one major correlate. Therefore, magnetic resonance imaging (MRI) scan has become one of the most common markers associated with cognitive scales performed in aging studies or in clinical practice to measure brain disease burden
[79]. Whilst hippocampal atrophy is an important imaging correlate of memory impairments observed in numerous brain disorders, its pattern of alteration may vary according to the disease and the stage of the disorder.
For instance, within hippocampal formation, the EC appears to be most resistant to the effects of normal aging, as changes are mainly restricted to the DG and CA3. In contrast, the EC is most vulnerable to AD while the DG and CA3 remain relatively preserved. With regard to the CA1 area and the subiculum, they are mainly affected in SCZ and MDD respectively. Unlike AD, no prominent cell loss has been identified in aging, SCZ and MDD, suggesting rather, functional alterations such as connectivity dysfunction
[79]. Consistently, an MRI-based study using diffusion tensor imaging to detect dendritic integrity revealed age-related alterations of DG and CA3 dendrites in aged patients
[80] (
Figure 2). Nevertheless, the measure of hippocampal volume was found to be sensitive enough to aging and to neurodegenerative and psychiatric disorders. For instance, after the age of 70, total hippocampal volume is believed to decrease at a rate of ~1.5% a year
[81]. Additionally, hippocampal volume loss has been shown to reach 10 to 15% in mild cognitive impaired (MCI) patients
[81]. Patients suffering from schizophrenia, PD or depression also exhibit hippocampal volume reduction of 4–6% relative to healthy subjects
[82][83][84].
Of most interest, several lines of evidence now support that 5-HT4Rs agonists could limit such hippocampal deterioration at different levels, notably in AD context.
First, the above reported hippocampal volume loss—either due to aging or pathological condition—can be compensated, at least partly, through neurogenesis boost, which is altered in various neurological and psychiatric diseases
[85]. However, it has been shown that sub-chronic treatment with 5-HT
4Rs agonists induced an increase in BDNF expression in the CA1 (72%) as well as in the DG (52%), this latter demonstrating a neuro-proliferative activity
[40]. Further, increased levels of other neurotrophic factors have also been reported after 5-HT
4Rs agonist treatment, such as the soluble (non-amyloidogenic) form of the amyloid precursor protein alpha (sAPPα) (
Figure 2). The functions of sAPPα include—but are not limited to—proliferation, neuroprotection, synaptic plasticity, memory formation, neurogenesis and neuritogenesis in cell culture and animal models. Quite interestingly, sAPPα production was found to be promoted following acute
[86] and chronic 5-HT
4Rs activation in various conditions that include cell lines overexpressing 5-HT
4Rs (50% increase)
[87][88][89][90][91] as well as neuroblastoma cell line
[87], and cultured neurons from a mouse model of AD
[55][92][93][94]. A similar effect was observed in vivo both in healthy mice (2-fold increase)
[86] and in AD mice models (1.5-fold increase)
[54][55]. In the context of AD, the effects of 5-HT
4Rs activation on sAPPα production would confer an additional benefit though a reduction in amyloid load (31–55% in a mouse model of AD
[54][55]) by limiting the amyloidogenic pathway. Indeed, accumulation of neurotoxic Aβ in key hippocampal regions appears to be the primary cause of neuronal death leading to hippocampal atrophy
[95] (
Figure 2).
Second, additional data supporting the putative role of 5-HT
4Rs in preserving hippocampal integrity come from studies focusing on dendritic spines hosting excitatory synapses. The latter are dynamic structures, whose formation, shape, volume and collapse depend on neural activity. Therefore, they influence (but also can in return be influenced) the learning processes and memory performance
[15]. In mice, pharmacological activation of 5-HT
4Rs was shown to selectively potentiate the learning-induced dendritic spines’ growth (+6%) within the hippocampal CA1 (
Figure 2). This was not found in other brain structures that are not as much implicated in memory processing (i.e., primary visual cortex)
[39]. Moreover, in a recent study using high resolution time lapse FRET imaging on neuronal dendrites, 5-HT
4Rs activation was found to prompt maturation of synaptic connections via the 5-HT
4R/G13/RhoA signaling cascade
[96]. By activating PKA and BDNF/TrkB signaling pathways, 5-HT
4Rs activation also promoted total dendritic length, number of primary dendrites and branching index in vitro
[97]. Since spines represent potential sites of postsynaptic excitatory input, boosting their growth and maturation may translate into an increase in the number of excitatory synapses.
Finally, it is worth mentioning that reactive astrocytes are found both in human AD patients and AD mice models. Post-mortem morphological brain studies demonstrate close interaction between astrocytes and Aβ deposition in AD patients. In fact, reactive astrocytes are thought to be involved in Aβ production by upregulating β-secretase activity and APP in the diseased brain
[98]. In this way, any strategy that would participate in a reduction in astrogliosis may substantially contribute to a reduction in Aβ load and subsequent neuronal loss. IL-1β and MCP-1 are two key pro-inflammatory mediators involved in glial reactivity whose levels have been found to be reduced by 30% to 45% following chronic 5-HT
4Rs activation in an early onset mouse model of AD
[54]. Consequently, astrogliosis and microgliosis were reduced by 50–60% and 57% respectively in the EC, an area of the hippocampal formation that is particularly susceptible to degeneration in AD, as previously discussed
[54][55]. Of note, astrogliosis reduction was even more pronounced with a longer duration of 5-HT
4Rs agonist treatment
[55]. Hence, 5-HT
4Rs modulation could modify AD pathogenesis by targeting inflammatory pathways in glial cells.
The demonstration of such beneficial effects of 5-HT4Rs ligands holds promise for the development of disease-modifying drugs, which represents a yet unmet medical need. Of course, upstream correction of the pathological drivers of the disease is crucial to significantly improving the downstream symptoms and to prevent progressive cognitive deterioration. To date, preclinical studies that showed beneficial effects of 5-HT4Rs on hippocampal function have been mainly performed in either non-pathological conditions or in experimental models of the disease (cell lines or animal models). However, it seems important to stress that the pathology of AD shares a number of hippocampal alterations with ageing, SCZ, MDD and PD as discussed above. This ultimately raises the hope for potential translation of such beneficial effects of 5-HT4Rs in a large number of brain diseases.
3.4. Functional Synaptic Plasticity Impairments
Considered as the cellular support of memory, LTP has received much attention in the search for a better understanding of the mechanisms involved in memory disorders. Veritably, impairment of hippocampal synaptic function is often considered as an early detectable feature of aging and/or pathological stage, well before the first memory symptom appearance or before the observation of hippocampal atrophy.
Downregulation of plasticity-related proteins such as cAMP and CREB have, for instance, been observed in the hippocampus of both animal models of AD, and AD patients
[99]. In this regard, there is accumulating evidence for a beneficial action of 5-HT
4Rs agonists on cAMP/CREB signaling. Consistently, increases in both cAMP and CREB levels as well as the phosphorylated form (active form) of CREB (pCREB) were found both in healthy rats
[40] and the neuroblastoma cell line
[93] following 5-HT
4Rs activation.
However, the effects of 5-HT4Rs modulation on synaptic plasticity have been little studied, with only eight studies performed between 2001 and present, and results varying according to the hippocampal subfield investigated (Table 1).
Table 1. Compilation of electrophysiological investigations of synaptic plasticity in rodents after pharmacological 5-HT4Rs activation.↑ denotes an increase; ↓ denotes a decrease; = denotes no change. Abbreviations: CA1, CA3: cornus ammonis 1,3; DG: dentate gyrus; DP: depotentiation; HFS: high frequency stimulation; LTD: long term depression; LTP: long term potentiation; LFS: low frequency stimulation; SUB: subiculum; TBS: theta burst stimulation.
Method
|
Hippocampal Area
|
Plasticity
|
Conditioning Stimulus
|
5-HT4Rs Agonist
|
Effects of 5-HT4Rs Activation on Plasticity
|
Reference
|
In vivo
|
DG
|
LTP
|
HFS (200 Hz)
|
RS67333
|
↓
|
Kulla and Manahan-Vaughan.2002
|
LTP
|
HFS (200 Hz)
|
5-Methoxytryptamine
|
=
|
|
LTP
|
HFS (10 × 400 Hz)
|
RS67333
|
Transient ↑ and curtailed
|
Marchetti et al. 2004
|
LTP
|
HFS (200 Hz)
|
RS67333
|
Curtailed
|
Twarkowski et al. 2016
|
DP
|
LFS (5 Hz)
|
RS67333
|
Blocked
|
|
LTD
|
LFS (1 Hz)
|
RS67633
|
↓
|
|
CA3
|
LTP
|
HFS (4 × 100 Hz)
|
RS67333
|
↓
|
Twarkowski et al. 2016
|
LTD
|
LFS (1 Hz)
|
RS67333
|
↓
|
CA1
|
LTP
|
HFS (5 × 400 Hz)
|
SC53116
|
↑
|
Matsumoto et al. 2001
|
LTP
|
HFS (4 × 100 Hz)
|
RS67333
|
=
|
Kemp and Manahan-Vaughan 2005
|
LTD
|
LFS (1 Hz)
|
RS67333
|
↓
|
Ex vivo
|
CA1
|
LTP
|
HFS (1 × 100 Hz)
|
RS67333
|
=
|
Lecouflet et al. 2020
|
LTP
|
TBS (4 × 5 Hz)
|
RS67333
|
↓
|
SUB
|
LTP
|
HFS (4 × 100 Hz)
|
RS67333
|
=
|
|
LTD
|
LFS (1 Hz)
|
RS67333
|
↑
|
Wawra et al. 2014
|
These data support the fact that 5-HT4Rs, through their modulatory effects on synaptic plasticity processes, will enable the hippocampus to ensure its filtering role of information during acquisition and more variable changes in the downstream areas.This perspective seems consistent with clinical data that suggest that an increased signal-to-noise ratio within the hippocampus improves the encoding accuracy, a function which is thought to be mainly supported by the DG where 5-HT4Rs are most abundantly expressed [100].
4. Concluding Remarks and Future Perspectives
Overall, arguments to consider 5-HT4Rs as a target of choice for the treatment of memory impairments mainly stem from preclinical evidence. In fact, only a few experiments were performed on humans.
Nonetheless, quite recently, SUVN-D4010, a novel, potent, highly selective 5-HT4Rs partial agonist intended for the treatment of cognitive disorders, was found to be safe and well tolerated in healthy human subjects, even in elderly population (Suven Life Sciences, NCT02575482 and NCT03031574). Lastly, the results published last year regarding prucalopride are also of high interest. Indeed, while already approved by the FDA in 2018 to treat chronic idiopathic constipation, prucalopride was investigated in a battery of cognitive tests related to hippocampal functions. In healthy human subjects, prucalopride showed beneficial effects on learning and memory performance (NCT03572790) [100] and is currently under investigation for its role in depression. Evidence for improved memory performance after 5-HT4Rs activation in humans was extended by a very recent fMRI study. Following prucalopride intake, hippocampal activity during memory recall was significantly increased compared with volunteers receiving a placebo [101].
The use of 5-HT4Rs ligands in the treatment of memory deficits is still an ongoing challenge but has long been—and still unfortunately is—restricted to AD and MDD. However, a number of functional and morphological changes within the hippocampus are a common denominator of a broader range of both normal ageing and neurological diseases (such as PD, MDD, SCZ). A large amount of data from both animal models and humans have now reached a consensus on the fact that 5-HT4Rs activation can attenuate some of these hippocampal dysfunctions. This ultimately raises the exciting potential of restoring—or at least limiting—memory decline in these pathologies. Nevertheless, a deeper understanding of the mechanisms at work is still needed and would help further development. In this view, studies that investigate 5-HT4Rs effects on hippocampal function in a more integrated view should provide substantial insights.