3.2 Pathological effects of the amylin system in AD

Several studies over the past two decades suggest that amylin contributes to the pathophysiology of AD. Numerous in vitro studies have shown that human amylin can be neuroinflammatory and toxic to cells, including neuronal [e.g., primary cultures of human fetal neurons (Jhamandas et al., 2011; Jhamandas and Mactavish, 2012) or rat fetal neurons (Lorenzo and Yankner, 1996; Tucker et al., 1998)] and glial cells [e.g., human astrocytoma cells (Gitter et al., 2000), primary cultures of hamster microglia (Colton et al., 2000)], particularly at high concentrations or when “aged” to enhance fibrillation and aggregation of the peptide. Given the similarities between these data and the neurotoxic effects of Aβ, this may suggest that amylin accumulation within the CNS of individuals with AD has detrimental effects similar to those produced by Aβ.

These findings lead to the question of the mechanisms by which amylin and Aβ exert neurotoxic effects in the CNS. Interestingly, it appears that amylin receptor signaling may mediate the effects of both peptides. In particular, the AMY3A receptor (CTR-A / RAMP3) appears to be relevant for AD (Fu et al., 2012; Jhamandas et al., 2011). It is thought that activation of AMY3A by Aβ or amylin exerts toxicity by altering potassium conductance (Jhamandas et al., 2011) and calcium concentrations (Fu et al., 2012; Kawahara et al., 2000) in the cell, followed by disruption of the cell membrane and initiation of apoptosis (Jhamandas and Mactavish, 2012). As the components of the AMY3A receptor are expressed in the brain (see Section 2.1), it is possible that these in vitro findings are recapitulated in vivo, and increased CNS AMY3A receptor signaling due to elevated amylin and/or Aβ may mediate some of the neurotoxic events of AD. Pharmacological antagonists of the amylin receptor can attenuate or block the neurotoxic effects of either amylin or Aβ (Jhamandas et al., 2011), suggesting the potential physiological relevance of this effect. Despite these findings, it should be noted that other groups have found that Aβ does not increase cAMP in HEK293S or Cos7 cells expressing AMY1A, AMY2A, or AMY3A, suggesting that Aβ may not activate amylin receptors (Gingell et al., 2014). There are several metholodogical differences between this study and others that may underlie the discrepant findings, including the use of cells transiently transfected with the amylin receptor (Gingell et al., 2014) versus the use of stably transfected cells (Fu et al., 2012) or human fetal neurons (Jhamandas et al., 2011; Jhamandas and Mactavish, 2012) as the model. Although this study brings into question whether the toxic effects of Aβ are fully amylin receptor-mediated, the majority of currently available data support the hypothesis that Aβ and amylin produce deleterious effects on cell function and survival via amylin receptors.

Recent data also indicate that increased levels of amylin can lead to its accumulation in the microvasculature of the brain, damaging both blood vessels and white matter in the HIP rat, a model in which human amylin is overexpressed; these rats are also hyperglycemic and exhibit some cognitive and behavioral deficits (Ly et al., 2017). However, in the same paper, amylin knockout rats given daily injections of aggregated human amylin for 1 week showed a slightly different pattern of effects; while amylin was still detected in the CNS microvasculature, white matter was not damaged (Ly et al., 2017). There are several differences in these animal models that might explain these different results; for example, chronically elevated amylin levels in the HIP rat could elicit compensatory changes not observed with the comparatively acute administration of amylin in the amylin knockout animal. Nevertheless, these results are consistent with human data indicating amylin accumulation in the CNS vasculature of patients with AD (Jackson et al., 2013) and may suggest another way in which amyloidogenic or aggregated amylin can potentially contribute to CNS damage.

3.3 Beneficial effects of amylin in AD

Based on the data discussed thus far, it would appear that amylin is strictly harmful or detrimental in AD. However, newer evidence suggests that the role of amylin in AD is not as straightforward as it may seem. Amylin or amylin-based pharmacotherapies have recently been shown to decrease levels of CNS amyloid in rodent models of AD. Indeed, systemic administration of amylin or the amylin analog pramlintide reduces the Aβ burden in areas of the brain that are heavily impacted by AD, such as the hippocampus and cortex, in the 5XFAD mouse model of AD (Wang et al., 2017; Zhu et al., 2015; Zhu et al., 2017b). Intriguingly, these reductions in central Aβ are thought to be driven by an efflux of Aβ from the brain to the blood. Intraperitoneal injection of amylin increased circulating levels of Aβ in the Tg2576 mouse (increases in both Aβ1–40 and Aβ1–42 in serum), the 5XFAD mouse (increased serum Aβ1–42), and the Dutch APP mouse (increased serum Aβ1–40) (Mohamed et al., 2017; Zhu et al., 2015). Of potential clinical relevance, similar effects were observed after subcutaneous pramlintide administration in human patients with AD, who displayed increased plasma Aβ1–40 after acute pramlintide treatment (Zhu et al., 2017a). The mechanisms by which amylin-induced efflux of Aβ occurs are not completely understood, but may involve LRP1, a protein involved in Aβ transport. In vitro studies in mouse brain endothelial cells, used as a model of the blood-brain barrier, show that amylin upregulates trafficking of LRP1 to the cell membrane (Mohamed et al., 2017) suggesting a potential mechanism by which amylin increases transport of Aβ from the CNS into the blood. In addition, although much of the research on amylin’s effects in AD has focused on reductions in CNS amyloid burden, there are a number of other neuropathological changes that occur in the brain in AD. Interestingly, amylin and amylin receptor agonists also appear to attenuate some of these other AD-related pathophysiologies, such as tauopathy and neuroinflammation, in various mouse models of AD including SAMP8 (Adler et al., 2014), 5XFAD (Wang et al., 2017; Zhu et al., 2017b), and 3xTgAD (Zhu et al., 2017b) mice.

Collectively, these data suggest that amylin treatment may actually be beneficial to treat AD-related pathophysiology. Given that one of the major symptoms of AD is cognitive decline, it is also critical to understand whether amylin influences memory and cognitive ability in AD, as amelioration of these symptoms would help to improve the quality of life for patients with AD. The majority of the available literature suggests that in general, plasma amylin is positively associated with cognitive ability. In elderly individuals, higher levels of amylin are associated with better performance on verbal and visuospatial memory tasks (Qiu et al., 2014), although this study did not account for AD diagnosis of the participants. Other studies have categorized participants by status of AD and/or mild cognitive impairment and show that individuals with AD or mild cognitive impairment have lower non-fasting plasma amylin levels compared to a cognitively normal control group (Adler et al., 2014), consistent with a positive correlation between amylin and cognition. However, another report shows that fasted amylin levels are higher in patients with mild cognitive impairment or AD compared to individuals without dementia (Morris et al., 2016). These discordant findings may be a result of fasting status at the time of blood draw, or could be due to the exclusion of individuals with moderate or severe AD in the latter study (Morris et al., 2016). Beneficial effects of amylin and amylin receptor agonists on cognitive ability are also revealed in animal models of AD. It has been consistently demonstrated that peripheral administration of amylin or pramlintide improves performance in tasks of learning and memory in various animal models of AD including SAMP8, 5XFAD, and 3xTgAD mice (Adler et al., 2014; Zhu et al., 2015; Zhu et al., 2017b). Importantly, the improvements in AD symptoms in 5XFAD mice produced by chronic amylin administration, including reduced amyloid burden and improved performance on learning and memory tasks (e.g., Y-maze) are attenuated by co-administration of AC253, suggesting that these benefits are mediated by amylin receptor activation (Zhu et al., 2017b). Collectively, these data indicate a beneficial effect of exogenous amylin / pramlintide administration on cognition and memory in humans with AD as well as in animal models of the disease.

Some studies suggest that amylin levels are lower in individuals with AD [(Adler et al., 2014; Qiu and Zhu, 2014); but also see (Fawver et al., 2014)]. If amylin is decreased in AD, it follows that at least some AD symptoms might be improved by restoring amylin signaling via administration of amylin or amylin analogs like pramlintide. Given the ability of amylin to cross-seed or associate with Aβ (Berhanu et al., 2013; Ono et al., 2014), one might expect that it would be critical to use nonamyloidogenic amylin to prevent further aggregation or plaque formation. However, human amylin, which is amyloidogenic and prone to aggregation, can produce reductions in central Aβ that are associated with improved cognitive ability in 5XFAD mice, as assessed by performance on the Y-maze and Morris water maze (Zhu et al., 2017b). The mechanism by which administration of exogenous amylin ligands – amyloidogenic or not – improves AD symptoms remains an open empirical question.

As noted throughout this section, the available in vivo results to date have been generated from a variety of animal models of AD. There are a number of key differences among these animal models, including the mutation(s) targeted as well as the particular physiological and behavioral symptoms of AD exhibited by each model [for review, see (Lee and Han, 2013; Platt et al., 2013; Webster et al., 2014)]. On one hand, the variety of AD models used to evaluate the effects of amylin and amylin analogs on the disease could be viewed as a limitation in the field, as it may reduce the ability to directly compare findings between studies. However, this could also be viewed as evidence in favor of the potential utility of amylin-based compounds to treat AD. Given that AD involves a constellation of pathophysiological and behavioral changes, and that in vivo studies generally support the idea that amylin and pramlintide produce beneficial effects on symptoms in several different mouse models of AD (Adler et al., 2014; Zhu et al., 2015; Zhu et al., 2017b), this may suggest broader applicability of amylin-based pharmacotherapies for treatment of this complex disease.

4.1 Amylin and its receptor: a complex system

As described in Section 3.3, administration of amylin receptor agonists (amylin, pramlintide) can attenuate symptoms of AD in animal models. However, a recent study suggests that treatment with an amylin receptor antagonist can also improve AD symptoms. Jhamandas’s group showed that central infusion of the amylin receptor antagonist AC253, or peripheral administration of cyclic AC253 (a modified form of AC253 with better CNS penetrance), improved performance on the Morris water maze in TgCRND8 mice (Soudy et al., 2017). This result is consistent with in vitro studies suggesting that amylin receptor antagonists block the toxic effects of amylin and Aβ (Fu et al., 2012; Jhamandas et al., 2011; Jhamandas and Mactavish, 2012), but also raises the question of how both agonists and antagonists of the amylin receptor can have beneficial effects on AD symptoms in animal models. This may relate to the complexities of amylin signaling, dosing of amylin receptor ligands, and the receptors to which amylin binds.

As described in Section 2.1, there are 6 possible amylin receptors that can be formed from the various combinations of CTRs and RAMPs. Amylin and amylin receptor agonists can bind to any of these receptors and engage downstream signaling events, but typically, research on amylin signaling in AD focuses on only one or a subset of the different amylin receptor subtypes. For example, Soudy et al. report that the amylin receptor antagonist cyclic AC253 is expected to potently inhibit AMY3A based on in vitro data, but its effects on other amylin receptors are not reported (Soudy et al., 2017). It has also been shown that AC253 may act as a partial agonist at the amylin receptor (Zhu et al., 2017b). Although this is untested for cyclic AC253, this could represent another mechanism by which amylin receptor activation could be differentially impacted by various ligands and perhaps provide some resolution to these discrepancies in the literature.

A related consideration is the dosing of the amylin receptor ligands in these and other studies. For example, several in vivo studies suggesting that pramlintide administration is beneficial in improving the symptoms of AD use chronic administration of pramlintide, with doses in the µg/kg range given daily for several weeks (Adler et al., 2014; Zhu et al., 2015). However, recently published in vitro electrophysiological analyses in mouse brain hippocampal tissue suggest that nanomolar concentrations of pramlintide can attenuate baseline long-term potentiation (LTP) deficits in the hippocampus of TgCRND8 mice as well as the reduction in LTP induced by application of amylin or Aβ, raising the possibility that pramlintide might act as an antagonist at the amylin receptor at low concentrations (Kimura et al., 2017). In addition, human amylin-induced changes in intracellular calcium in rat hippocampal neurons in vitro were mediated by amylin receptors at low concentrations of amylin, but at higher concentrations the effects were independent of amylin receptors, possibly due to aggregation of the ligand at higher concentrations (Zhang et al., 2017b). These findings suggest that dosing may be a critical factor in teasing apart the beneficial versus detrimental effects of amylin receptor ligands on features of AD. Unfortunately, much of the available in vivo work does not include thorough physiological and behavioral dose-response analyses that would be required to determine whether the differential effects of various doses of amylin receptor ligands in vitro are recapitulated in vivo. This is especially important given the interest in amylin-based compounds as potential pharmacotherapies for treating AD; although the literature clearly shows that certain doses of amylin and pramlintide can improve AD symptoms, it is unclear what the effective ranges may be. This is a critical area of investigation that needs to be addressed in the literature. Furthermore, it is also important to gain a better understanding of the dose-response effects of these ligands not only on their own, but also to evaluate whether their function may be different in the presence of other amylin receptor ligands, which may shed light onto some of the existing discrepancies between current in vivo and in vitro data.

It is worth noting that amylin or pramlintide may also change the activity of receptors other than amylin receptors. Amylin belongs to a family of peptides including calcitonin, calcitonin gene-related peptide (CGRP), and adrenomedullin that are highly promiscuous and can bind to several types of receptors with differing binding affinity (Hay et al., 2006; Poyner et al., 2002; Wimalawansa, 1997). In addition to binding to amylin receptors, amylin can bind to the naked CTR (e.g., without an associated RAMP) as well as the calcitonin receptor-like receptor (CLR) with or without RAMP (Hay et al., 2006). Therefore, amylin receptor ligands are capable of altering signaling at a variety of receptors which could potentially have different consequences for AD. “Off-target” (e.g. non-amylin receptor- / non-AMY3A-mediated) binding of an amylin receptor agonist or antagonist could change the net effects of amylin signaling in the brain in a way that improves AD symptoms, highlighting the need to carefully and systematically evaluate the role of various amylin and CGRP-family receptors in the effects of amylin-based ligands on AD.

4.2 Age-related changes in the amylin system

In thinking about the potential utility of amylin-based pharmacotherapies for the treatment of AD in humans, the effect of aging on the amylin system has often been overlooked. Our understanding of changes to the amylin system during healthy aging or in a pathophysiological state such as AD is extremely limited. A few reports describe age-related changes to the amylin system, and of those, most have examined plasma amylin levels. While some studies suggest that there is no significant association between age and plasma amylin levels in older adults (Li et al., 2016a), other reports comparing younger and older adults have revealed that amylin release is decreased in older adults (Dechenes et al., 1998) or in middle age but not older adulthood (Edwards et al., 1996). The reason for these discrepant results is unknown but may be related to methodological differences among these studies.

Another important question is whether the physiological effects of amylin are the same throughout the lifespan or whether they differ in aging. Food intake studies in rats demonstrate that food-deprived older rats display an anorectic response to amylin at a dose that does not impact feeding in young rats that have been deprived of food for the same amount of time (Lutz et al., 1994). Although this difference could be due to changes in baseline energy balance control in older rats (Lutz et al., 1994), this still suggests that aging may directly or indirectly impact the efficacy of amylin signaling and/or function. In order to critically evaluate the potential clinical relevance of amylin-based pharmacotherapies for AD, it is important to understand not only what happens to the amylin system during normal aging but also whether AD causes further changes in expression or function of the amylin system. Potential mechanisms underlying age-and/or AD-related changes in amylin function may include alterations in amylin receptor expression, intracellular response to receptor agonists, amylin release (as described above), or another aspect of amylin physiology. An age-related increase in CTR and RAMP3 expression has been observed in discrete brain areas of the TgCRND8 mouse model of AD (Jhamandas et al., 2011), supporting the idea that AD may change amylin signaling and highlighting the need to examine this more broadly. Yet in general, these possibilities are extremely underinvestigated.

Understanding the amylin system in aging and deciphering how it may relate to AD is further complicated by needing to understand how expression of amylin and its receptors is altered not only during normal healthy aging versus AD, but also in AD-related comorbidities such as T2DM and obesity. These represent important areas of investigation that will provide a more comprehensive understanding of amylin physiology required to fully understand the effects of the peptide in AD. Clearly there are several key gaps in our current knowledge of how the amylin system changes over the lifespan, even within the context of normal healthy aging. Moreover, diseases such as T2DM and obesity are often comorbid with AD and are known to influence the physiology of the amylin system (Johnson et al., 1989; Pieber et al., 1994; Reinehr et al., 2007). More research is undoubtedly required to understand the basic physiological changes that occur in the amylin system during aging, as well as how AD and related comorbidities may change the system and potentially alter the efficacy of amylin-based pharmacotherapies.

The author thanks Katherine Balantekin for her helpful editorial suggestions.

Funding

This work was supported by the National Institutes of Health [grant numbers DK103804, DK114211].

The author receives funding from Zealand Pharma that was not used in support of this work.