The Mann–Whitney U test showed that there were no significant differences in training latencies displayed by the 1.0 mA TTX (40.20 s) and VEH (26.20 s) groups (p = 0.18), as well as in the 3.0 mA TTX (28.80 s) and VEH (26.40 s) groups (p = 1.0). Similarly, there were no significant differences in escape latencies between the TTX and VEH groups, regardless of the foot-shock intensities. Median escape latencies displayed by the 1.0 mA groups were 1.80 and 1.40 s, respectively (p = 0.49), and median escape latencies of the TTX and VEH groups trained with 3.0 mA were 0.80 and 0.60 s, respectively (p = 0.27) (data not shown).

#### Long-Term Memory

The TTX group trained with 1.0 mA displayed a significantly lower score than its VEH control group (p < 0.05) during the 48-h retention test. In contrast, no differences were found when comparing the TTX and VEH groups trained with 3.0 mA (p = 0.13) (**Figure 2A**).

#### Acquisition

To evaluate whether the amnestic effect of pre-training infusion of TTX in the 1.0 mA PL group was due to interference with learning of the IA task rather than with consolidation, TTX or VEH was administered 25 min before training with 1.0 mA, and

retention was measured twice: at 30 min and at 48 h after training. The results showed no significant differences between the TTX and VEH groups on the retention test run 30 min after training (p = 0.95) while, again, a reliable deficit was shown by the TTX group in the 48-h test (p < 0.05) (**Figure 2B**).

#### State Dependency

Because the TTX was administered 25 min before training, and retention was measured 48 h later in a non-drug state, it was feasible that the amnesia thus produced could have been due to a state-dependent effect. To rule out this possibility, two groups of rats were treated twice, with either TTX or VEH, 25 min before training and 25 min before retention testing. In comparison to the VEH group, the TTX group showed reliable amnesia (p < 0.05) (**Figure 2C**).

#### c-Fos Immunohistochemistry

Tetrodotoxin administration into PL induced a significant decrement in c-Fos expression relative to VEH in the groups that had been trained with 1.0 or 3.0 mA (p < 0.05, in each case) (**Figure 2D**).

As mentioned in Section "Materials and Methods," c-Fos expression was also measured within a 100 µm × 100 µm counting box located 600 µm below the PL injector-tip tracks. We found that TTX did not interfere with c-Fos expression, as there were no significant differences between the VEH and TTX groups, p = 0.73 (**Figure 2E**). This finding demonstrates that the deficit in memory consolidation seen in the animals that had been trained with 1.0 mA was due to inactivation of PL and not to diffusion of the drug into the more ventrally located IL.

**Figure 2F** is a representative photomicrograph showing placement of cannula tip in PL.

#### Infralimbic Cortex

#### Training and Escape Latencies

The Mann–Whitney U test showed that there were no significant differences in training latencies between the TTX and VEH groups, regardless of the foot-shock intensities. Median training latencies displayed by the 1.0 mA groups were 33.60 and 25.20 s (p = 0.80), respectively. In the VEH and TTX groups trained with 3.0 mA, training latencies were 24.20 and 23.50 s (p = 0.97), respectively (data not shown). Similarly, there were no significant

differences in escape latencies between the TTX and VEH groups, regardless of the foot-shock intensities. Median escape latencies displayed by the 1.0 mA groups were 1.70 and 1.20 s (p = 0.28), respectively. In the VEH and TTX groups trained with 3.0 mA, escape latencies were 2.70 and 2.30 s (p = 0.65), respectively (data not shown).

As in the case of PL, TTX infusion into IL produced a significant retention deficit during the 48-h post-training session in the group that had been trained with 1.0 mA (p < 0.01 vs. VEH). Unexpectedly, TTX produced the same amnestic effect in the 3.0 mA group (p < 0.005 vs. VEH) (**Figure 3A**).

#### Acquisition

To evaluate whether the amnestic effect of pre-training infusion of TTX had been due to interference with learning rather than with consolidation, two groups of rats were trained with the low foot-shock (1.0 mA) and subjected to TTX or VEH injections into the IL 25 min before training. Retention was measured twice: at 30 min and at 48 h after training. The results showed no significant differences between the TTX and VEH groups during the retention test run 30 min after training (p = 0.85), while a reliable deficit was shown by the TTX group in the 48-h test (p < 0.02) (**Figure 3B**).

#### State Dependency

To determine whether the amnestic effect of pre-training TTX seen in the 1.0 and 3.0 mA IL groups during the 48-h retention test (**Figure 3A**) might have been due to state-dependency, two groups of rats were trained with 1.0 mA and another two groups were trained with 3.0 mA. Half of each group was treated twice with TTX and the other half with VEH, also twice, 25 min before training and 25 min before retention testing. TTX produced a significantly lower retention score relative to its VEH control group after training with 1.0 mA group (p < 0.05). In contrast, state-dependency was produced when 3.0 mA was used for training, as there were no reliable differences in retention scores between the TTX and VEH groups that had been trained with 3.0 mA (p = 0.11) (**Figure 3C**).

#### Post-training TTX Administration into IL

The results of the preceding experiment showed that pre-training TTX infusion into the IL produced a clear state-dependent effect when training was conducted with 3.0 mA, but not when 1.0 mA was used. Thus, the apparent amnestic effect produced after a single pre-training TTX infusion (**Figure 3A**) could be explained by the interaction of the high foot-shock and the differential pharmacological state of the IL cortex during training (drugged

state) and retention testing (non-drugged state). This outcome did not allow us to answer the question of whether IL has a role in memory consolidation when a high aversive stimulation is used to produce learning. To shed light into this matter, we decided to study the effects of IL inactivation with TTX induced after training, thus avoiding the confounding effect of statedependency. To this end, two groups of rats were trained with 1.0 mA and another two groups were trained with 3.0 mA. Half of each group was treated with TTX and the other half with VEH. The infusions were made immediately after training. A significant retention deficit was observed in the TTX group that had been trained with 1.0 mA (p < 0.03 vs. VEH), whereas no significant differences between the TTX and VEH groups trained with 3.0 mA were found (p = 0.11) (**Figure 3D**).

#### c-FOS Immunohistochemistry

The infusion of TTX into the IL induced a significant decrement in c-Fos expression relative to VEH in the groups that had been trained with 1.0 or 3.0 mA (p < 0.05 for each comparison) (**Figure 3E**).

## DISCUSSION

The main findings of this study, where TTX was administered before training, were that regardless of the intensity of training, transient inactivation of ACC did not disrupt memory consolidation of the IA task. In contrast, in PL and IL TTX produced a highly significant deficit of consolidation when the moderate foot-shock was used in training. Interestingly, the retention deficit was still evident after training with the high foot-shock when IL had been inactivated, due to statedependency, but retention was not diminished in the PL group (**Figures 1A**, **2A**, **3A**). When TTX was administered immediately post-training into IL, it interfered with consolidation only when the moderate foot-shock was used. We suggest that these differential effects are dependent on the dissimilar connectivity of the three regions that were studied. They receive strong connections from the same thalamic regions; PL and IL receive afferents from the basolateral and basomedial nuclei of the amygdala; and PL is more densely connected to limbic cortical areas than ACC and IL (Hoover and Vertes, 2007). Further research is needed to study the contribution of these different anatomical interactions in memory consolidation of IA.

The histochemical results showed that administration of TTX in each of those cortical regions produced reliable neuronal inactivation, as evidenced by the diminished detection of c-Fos near the injector tips. Because there were no significant differences in training and escape latencies between the TTX- and VEH-treated animals, irrespective of the microinjected region or the intensity of the foot-shock used for training, the impaired retention that was found in the PL and IL groups cannot be explained by any potential deficiency of the motor or perceptual activities necessary to perform the IA task. In other words, the treated animals could cross from the safe compartment to the shock compartment, and escape from the foot-shock just as efficiently as the VEH-treated animals. What follows is a discussion focusing on relevant studies on IA.

#### Anterior Cingulate Cortex

Inactivation of ACC did not interfere with memory consolidation of IA, as indicated by the high retention scores of animals trained with 1.0 and 3.0 mA. This agrees with the report of Mello e Souza et al. (1999) that intra-ACC administration of muscimol and AP5 did not impede the formation of longterm memory of this task. Our result is also congruent with the lack of impairment of memory consolidation of IA found after pre-training radiofrequency lesion (Chai et al., 2010) of ACC. Taken together, these data suggest that this region is not involved in neural activity encoding needed for memory consolidation of the CS-UCS association during training of the IA task. This interpretation must be taken cautiously, because other lines of research suggest that ACC is involved in memory consolidation of IA. Thus, infusion of the cholinergic agonist oxotremorine into ACC immediately after IA training improved memory (Malin and McGaugh, 2006) and, consistent with this finding, it was shown that pre-training and posttraining infusion of scopolamine, a cholinergic antagonist, impaired memory consolidation of this task (Riekkinen et al., 1995). Furthermore, administration of a protein synthesis inhibitor into ACC or mPFC (which included PL and IL) produced a significant retention deficit of IA (Zhang et al., 2011). New studies are needed to comprehend these dissimilar results.

## Prelimbic Cortex

The findings that inactivation of PL produced a marked deficiency of retention when the low intensity foot-shock was used for training, and that it did not impede performance when the high foot-shock was used (**Figure 2A**) fit well with previous results where interference with neural activity of striatum (Giordano and Prado-Alcalá, 1986; Pérez-Ruiz and Prado-Alcalá, 1989), hippocampus (Quiroz et al., 2003; Garín-Aguilar et al., 2014), amygdala (Parent et al., 1992, 1994), and substantia nigra (Cobos-Zapiaín et al., 1996; Salado-Castillo et al., 2011) disrupted memory consolidation when IA training took place with a low intensity of aversive stimulation, but not when stimulation of relatively high intensity was used.

That the impairment in retention shown by the TTX group that had been trained with the low foot-shock was due to interference with memory consolidation, and not to a deficiency in learning, was demonstrated by the optimal performance shown by the group of animals that was tested 30 min after the administration of the drug. A deficit in consolidation became evident when this group was given a second retention test 48 h later (**Figure 2B**).

Because training took place under the influence of TTX, and retention of the task was measured when the animals were in a non-drugged condition, the possibility existed that the retention deficit observed in the low foot-shock group was due to a phenomenon of state-dependency and not to disturbance of memory consolidation. This possibility was

discarded because a group of rats that was trained and tested under the same pharmacological condition exhibited a deficient retention (**Figure 2C**).

The retention deficit observed in the present study gives support to the findings of Santos-Anderson and Routtenberg (1976) and of Jinks and McGregor (1997). The former authors showed that low-level electrical stimulation of the ventral aspect of the mPFC interfered with memory consolidation of IA, and the latter found that electrolytic lesions of PL produced a deficit in IA. Together, these findings indicate that PL has similar functions to those of the striatum, hippocampus, amygdala, and substantia nigra regarding memory consolidation of IA, i.e., these structures are necessary for memory consolidation under conditions of moderate training because interference with neural activity of any one of them impedes the formation of long-term memory. On the other hand, consolidation takes place after intense training despite this interference. It has been hypothesized that these structures are not critical for mediating associative processes derived from intense training, which produces plastic changes allowing for the recruitment of other structures.

#### Infralimbic Cortex

The study of IL yielded a complex set of data. In agreement with previous results, where lesions of IL produced a significant retention deficit of IA (Jinks and McGregor, 1997), we found that infusion of TTX into IL had the same detrimental effect when the low foot-shock was used for training. Contrary to our expectations, intense training did not protect memory consolidation against the inactivation produced by the TTX (**Figure 3A**). The memory test that was made 30 min posttraining to the group of animals that were treated with TTX into IL yielded top retention scores, while a reliable deficit was observed 48 h later in this same group. As in the case of PL inactivation, these results indicate that learning took place and that the impaired retention was due to a failure in memory consolidation (**Figure 3B**).

When TTX was infused into IL twice (both before training and before the 48-h retention test) a retention deficit was produced in the low foot-shock group but, unexpectedly, not in the high foot-shock group (**Figure 3C**). To the best of our knowledge, this is the first time that a state-dependent effect has been found in mPFC. Thus, the question of whether IL is involved in memory consolidation of intense training could not be answered with this experimental design. This problem was solved by administering the treatments immediately after training with the low and the

### REFERENCES

high intensity of foot-shock, thus avoiding the induction of statedependency. This manipulation confirmed that inactivation of IL impedes memory consolidation when IA training is carried out with an aversive stimulus of low intensity, and it revealed that, indeed, intense training protects against the amnestic effect of inactivation produced by the TTX (**Figure 3D**).

# CONCLUSION

The data obtained in this experimental series indicate that (a) memory consolidation of IA is not dependent on neural activity of the ACC; (b) normal activity of PL is essential for memory consolidation of moderate IA training, but not for acquisition or for consolidation of intense training; (c) normal activity of IL is also essential for memory consolidation of moderate IA training but not for acquisition or for consolidation of intense training. Moreover, the combined effect of TTX and intense training induces state-dependency.

# AUTHOR CONTRIBUTIONS

Designed the experiments: MT-G, AM, and RP-A. Performed the behavioral and histological experiments: MT-G and AM. Analyzed the data: MT-G and AM. Wrote and provided comments and discussion for the manuscript: MT-G, AM, GQ, and RP-A.

# FUNDING

This research was supported by Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (UNAM; IN201415) and by Consejo Nacional de Ciencia y Tecnología (CONACYT; 237570). MT-G is a doctoral student from Programa de Doctorado en Ciencias Biomédicas, UNAM and received fellowship 245656 from CONACYT.

### ACKNOWLEDGMENTS

The authors thank Bertha Islas, Norma Serafín, Omar González, Leonor Casanova, Martín García, and Sandra Hernández for their excellent technical and administrative assistance. We also wish to thank Jessica González Norris for editorial comments.

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cortex for the consolidation of inhibitory avoidance memory. Mol. Brain 4:4. doi: 10.1186/1756-6606-4-4

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Torres-García, Medina, Quirarte and Prado-Alcalá. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Potential Therapeutic Effects of Lipoic Acid on Memory Deficits Related to Aging and Neurodegeneration

Patrícia Molz 1, 2 and Nadja Schröder 1, 2 \*

1 Graduate Program in Medicine and Health Sciences, Faculty of Medicine, Pontifical Catholic University, Porto Alegre, Brazil, 2 Neurobiology and Developmental Biology Laboratory, Faculty of Biosciences, Pontifical Catholic University, Porto Alegre, Brazil

The aging process comprises a series of organic alterations, affecting multiple systems, including the nervous system. Aging has been considered the main risk factor for the advance of neurodegenerative diseases, many of which are accompanied by cognitive impairment. Aged individuals show cognitive decline, which has been associated with oxidative stress, as well as mitochondrial, and consequently energetic failure. Lipoic acid (LA), a natural compound present in food and used as a dietary supplement, has been considered a promising agent for the treatment and/or prevention of neurodegenerative disorders. In spite of a number of preclinical studies showing beneficial effects of LA in memory functioning, and pointing to its neuroprotective potential effect, to date only a few studies have examined its effects in humans. Investigations performed in animal models of memory loss associated to aging and neurodegenerative disorders have shown that LA improves memory in a variety of behavioral paradigms. Moreover, cell and molecular mechanisms underlying LA effects have also been investigated. Accordingly, LA displays antioxidant, antiapoptotic, and anti-inflammatory properties in both in vivo and in vitro studies. In addition, it has been shown that LA reverses age-associated loss of neurotransmitters and their receptors, which can underlie its effects on cognitive functions. The present review article aimed at summarizing and discussing the main studies investigating the effects of LA on cognition as well as its cell and molecular effects, in order to improve the understanding of the therapeutic potential of LA on memory loss during aging and in patients suffering from neurodegenerative disorders, supporting the development of clinical trials with LA.

Keywords: memory, lipoic acid, neuroprotection, aging, neurodegenerative disorders

# INTRODUCTION

Aging is a multifactorial process that involves genetics, lifestyle, and environmental factors (Hagen et al., 1999; Savitha and Panneerselvam, 2006; Lopez-Otin et al., 2013; Kennedy et al., 2014). During aging, biological processes promote the gradual loss of the individual's ability to maintain homeostasis, followed by a progressive deterioration in biochemical and physiological functions of the organism, increasing the susceptibility to diseases associated with aging (Arivazhagan et al., 2001a; Kumaran et al., 2005; Singh et al., 2015). Cognitive function also declines with age (Liu, 2008).

#### Edited by:

Antonella Gasbarri, University of L'Aquila, Italy

#### Reviewed by:

Enrique Cadenas, University of Southern California, United States Cedric Williams, University of Virginia, United States

> \*Correspondence: Nadja Schröder<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology

Received: 26 June 2017 Accepted: 06 November 2017 Published: 12 December 2017

#### Citation:

Molz P and Schröder N (2017) Potential Therapeutic Effects of Lipoic Acid on Memory Deficits Related to Aging and Neurodegeneration. Front. Pharmacol. 8:849. doi: 10.3389/fphar.2017.00849 Molz and Schröder Lipoic Acid and Memory

Aging has been associated to a more oxidized state in the redox balance (Jones and Sies, 2015), and the central nervous system becomes vulnerable to oxidative stress (Arivazhagan et al., 2002; Kidd, 2005; Ferreira et al., 2013; Zuo and Motherwell, 2013), defined as an unbalanced redox signaling, related to increased amounts of oxidants and ineffective antioxidant defenses (Go and Jones, 2017). In this context, nutrition can be considered a critical life-style factor that impacts the development and progression of neurodegenerative diseases (Virmani et al., 2013). Dietary supplementation with mitochondrial nutrients could promote natural neuroprotective effects, delaying the onset or progression of cognitive dysfunction and neurodegenerative diseases (Miquel, 2002; Abadi et al., 2013; Di Domenico et al., 2015; Mehrotra et al., 2015).

Over the years, lipoic acid (LA) has received increased attention as a nutritional supplement with therapeutic potential in the treatment or prevention of different pathologies (Shay et al., 2009; Rochette et al., 2013; Park et al., 2014), such as neurodegenerative diseases (Packer et al., 1997; Hager et al., 2001; Holmquist et al., 2007; Farr et al., 2012). LA has been shown to improve mitochondrial function (Kidd, 2005; Zhang et al., 2010; Zuo and Motherwell, 2013; Hiller et al., 2016), in addition to protect from cognitive dysfunction associated to aging and neurodegenerative diseases (Hager et al., 2007; Moreira et al., 2007; Liu, 2008). Thus, this study aims to review and discuss main findings showing potential memory-improving effects, as well as neuroprotective effects of LA, giving support to its use as an adjuvant in the treatment of neurodegenerative disorders. For this, we focused in discussing experimental studies addressing behavioral evaluations and cellular and molecular effects of LA.

# LIPOIC ACID

LA (1,2-dithiolane-3-pentonoico acid, thioctic acid) (Shay et al., 2009) was discovered in 1937 by Snell et al. (1937) and characterized by Reed et al. (1951). LA has been considered a powerful micronutrient presenting a range of pharmacological properties (Rochette et al., 2013; Koufaki, 2014; Park et al., 2014); however, many aspects of LA effects still need to be clarified, especially its application in the treatment and prevention of neurodegeneration.

# Chemistry of LA

LA is a low molecular weight dithiol with a chiral center containing eight-carbon disulfide in its structure (**Figure 1**; De Araujo et al., 2011). It is naturally occurring in all prokaryotic and eukaryotic cells (Bast and Haenen, 2003; Park et al., 2014). LA acts as co-factor in multienzyme complexes in the mitochondria, such as pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (Holmquist et al., 2007; Ghibu et al., 2009). A substantial part of LA is reduced to dihydrolipoic acid (DHLA) by lipoamide dehydrogenase (E3 component of the pyruvate dehydrogenase complex and α-ketoglutarate dehydrogenase) with involvement of the NADH and NADPH system (Arivazhagan et al., 2001b; Bilska et al., 2007). Reduction of LA to DHLA may also be completed by other cellular reducing systems, including

NAD(P)H-driven enzymes, such as thioredoxin reductases (Rochette et al., 2013). LA also contains an asymmetric carbon resulting in two optical isomers, the S form and the R form, with the former being synthesized endogenously (De Araujo et al., 2011).

### Dietary Sources, Metabolism, Toxicity, and Nutritional Recommendations

LA may be obtained from the diet (Packer et al., 1997; Hagen et al., 1999; Morikawa et al., 2001; Ghibu et al., 2009) and due to its capability of endogenous synthesis it is not considered a vitamin (Packer et al., 1995, 2001; Bast and Haenen, 2003), but is structurally considered a member of the vitamin B family (Xing et al., 2015). When ingested as a nutritional supplement preferably in the racemic mixture form, LA contains two isomers (1:1 of R-LA and S-LA), in which S-LA may prevent polymerization of R-LA and thus increases the bioavailability of the later (De Araujo et al., 2011).

LA is found in both vegetable and animal-based foods, identified as lipoyl-lysine (coupling of LA to specific lysine residues; Ghibu et al., 2009; Shay et al., 2009; Rochette et al., 2013; Li et al., 2015). R-LA is most abundantly found in vegetables such as spinach, broccolis, and tomatoes, which contain, respectively, 3.15 ± 1.11, 0.94 ± 0.25, and 0.56 ± 0.23 × 10−3 g lipoyl-lysine (gram dry weight). In foods of animal origin, the highest amounts of lipoyl-lysine are found in bovine kidney, heart, and liver, containing, respectively, 2.64 ± 1.23, 1.51 ± 0.75, and 0.86 ± 0.33 × 10−3 g lipoyl-lysine (gram dry weight) (Lodge and Packer, 1999).

The biochemistry and pharmacokinetics of LA have been extensively reviewed elsewhere (Packer et al., 1995; Bustamante et al., 1998; Cremer et al., 2006b; Shay et al., 2009; Rochette et al., 2013). In summary, either from food or nutritional supplement sources, LA is rapidly absorbed, metabolized and excreted (Moini et al., 2002; Rochette et al., 2013). LA is absorbed rapidly from the gastrointestinal tract (Hagen et al., 1999; Jalali-Nadoushan and Roghani, 2013), up to 93% of a dose administered orally is absorbed in the gastrointestinal tract, (Cremer et al., 2006b) and is clearly subject to considerable pre-systemic elimination (Hagen et al., 1999). Approximately 27–34% LA orally administered is available for absorption by the tissues (Hagen et al., 1999) and the liver is one of the main clearance organs because it has a high absorption and storage capacity (Bustamante et al., 1998; Hagen et al., 1999).

Studies have shown that the gastrointestinal absorption of LA is highly variable and its efficiency appears to be reduced when ingested in the diet, suggesting that the absorption of LA competes with other nutrients (Packer et al., 2001; Shay et al., 2009; Rochette et al., 2013). Gastrointestinal uptake of LA is fast and its presence in the plasma is followed by a fast clearance (Shay et al., 2009). The plasmatic half-life of LA is 30 min. Endogenous plasma levels of LA and DHLA in humans are, respectively, 1–25 × 10−9 g/mL and 30–140 × 10−9 g/mL (Ghibu et al., 2009; Rochette et al., 2013). Urinary excretion is maximal 3–6 h following LA administration. Approximately 45% is excreted in the urine during the first 24 h and only 3% is excreted in the feces. Moreover, only a small amount of the administered LA is excreted in the unaltered form (Bustamante et al., 1998).

There are no recommendations for daily LA intake in humans. However, the safety of LA was determined in acute and subchronic toxicity studies as well as its mutagenicity/genotoxicity in in vitro and in vivo studies. In dogs, a LD50 of 400–500 mg LA/kg b.w. has been reported (Packer et al., 1995). In rats a LD50 of 2,000 mg/kg b.w. was described, with some rats presenting signs of reduced well-being, including sedation, apathy, piloerection, hunched posture, and/or eye closure (Cremer et al., 2006b). On the other hand, studies evaluating oral LA supplementation up to 60 mg/kg per day in rats showed no adverse effects concerning body weight, histopathological findings, and blood analyses (Cremer et al., 2006a,b). Therefore a NOAEL (no observed adverse effect level) for rats of 60 mg/kg/day for longterm LA supplementation was provided. Clinical trials using LA to assess adverse health effects in humans were performed in doses up to 2,400 mg/day with no reported adverse effects vs. placebo (Shay et al., 2009). In spite of that, the exact doses that could induce adverse human health effect are still to be set up.

 Neurodegeneration is characterized by its insidious and chronic progressive onset and aging has been considered the main risk factor (Lin and Beal, 2006; Chen et al., 2012; Virmani et al., 2013; Irwin et al., 2016).

During aging, deleterious changes accumulate, causing the gradual decline of the biochemical and physiological functions (Arivazhagan and Panneerselvam, 2002; Kumaran et al., 2005; Santos et al., 2013).

Over the last decades, a wide range of studies have shown that progression of neurodegeneration is associated with increased DNA damage (partly is attributed to an imbalance between antioxidant and prooxidant factors; Chen et al., 2012; Kim et al., 2015). In addition, mitochondrial decline, leading to cognitive dysfunction (Liu, 2008; Aliev et al., 2009; Bishop et al., 2010; Irwin et al., 2016) as well as aggregation of oxidized proteins, accumulation of metals, inflammation and excitotoxicity have been reported (Moreira et al., 2007). Oxidative stress and mitochondrial dysfunction are interrelated mechanisms that play a central role in aging brain (Santos et al., 2013), since the continuous generation of reactive oxygen species (ROS), mainly the superoxide anion (O−• 2 ) at complexes I and III of the mitochondrial respiratory chain, throughout life produces a sustained oxidative stress by aging, possibly resulting in cognitive impairments (Limoli et al., 2004; Kumaran et al., 2005; Bagh et al., 2011). Although the exact mechanisms underlying the effects of unbalanced redox signaling are not completely elucidated, studies suggest that it plays a role in the pathogenesis of neurodegenerative disorders (Liu et al., 2017).

Brain aging has been related to structural alterations and inflammation, accompanied by cognitive and memory dysfunctions (Bagh et al., 2011; Pizza et al., 2011; Thakurta et al., 2014). With the aging of the world population and the increasing life expectancy, the risk of developing neurodegenerative diseases is higher than ever, as they are affecting millions of people each year in epidemic proportions (Santos et al., 2013; Irwin et al., 2016). As a result, an inevitable socioeconomic burden on our health care systems will occur. Effective prophylactic and therapeutic treatments are urgent for this group of seemingly inexorable diseases. In this review we will focus on discussing the ameliorating effects of LA on cognitive deficits observed in animal models of aging and neurodegenerative disorders.

# EFFECTS OF LA IN EXPERIMENTAL MODELS OF MEMORY DEFICITS ASSOCIATED TO NEURODEGENERATIVE DISORDERS

### Behavioral Studies

#### Alzheimer's Disease

Alzheimer's disease (AD) is the most common neurodegenerative disorder that causes dementia and affects middle to old-aged individuals (Hager et al., 2001; Gonzalez et al., 2014; Irwin et al., 2016). AD is characterized by progressive loss of cognitive functions, including memory, language, and reasoning (Di Domenico et al., 2015).

Studies have investigated the effects of LA in experimental AD models (Jesudason et al., 2005; Siedlak et al., 2009; Ahmed, 2012; Sancheti et al., 2013). For instance, Quinn et al. (2007) evaluated the chronic dietary supplementation with LA on hippocampusdependent memory of aged Tg2576 mice, a transgenic model of cerebral amyloidosis associated with AD. LA treatment was shown to reduce hippocampal-dependent memory deficits, significantly improving learning and memory in the Morris water maze in comparison to Tg2576 mice that did not receive LA. However, no significant differences in β-amyloid levels were found between Tg2576 mice that received LA in comparison to the ones that did not receive LA, indicating that chronic LA supplementation in the diet can ameliorate hippocampal memory impairments in Tg2576 mice without any effect on β-amyloid levels or plaque deposition.

Another study assessed the effects of LA in senescenceaccelerated mouse prone 8 (SAMP8) mice, associated to learning and memory impairments, and showed that LA can improve memory, in different paradigms (Farr et al., 2012). In object recognition, results indicated that mice that received LA presented a higher memory index than vehicle-treated mice. When memory was tested in the Barnes maze, results indicated that LA-treated mice spent more time by the target, and made fewer errors than controls, but did not present differences in speed in traversing the maze (distance/time) during training.

On the other hand, Siedlak et al. (2009) investigated young and aged mice overexpressing amyloid-β precursor protein (APP) and controls and showed that administration of R-LA had little effect on Y-maze performance. The authors concluded that, although oxidative stress has been proposed to mediate amyloid pathology and cognitive decline in aging, long-term LA administered within tolerable nutritional levels, presented limited benefit.

#### Parkinson's Disease

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in aging individuals and features motor symptoms related to dopaminergic neuronal loss in the substantia nigra, which results in decreased striatal dopaminergic terminals (Beal, 2003; De Araujo et al., 2011). Studies have shown that, in addition to rescuing cognitive deficits, LA is also able to ameliorate motor impairment related to PD. The effects of LA were examined in a rat model of PD induced by rotenone. The effect of LA (50 mg/kg/day, p.o.) was evaluated after the administration of rotenone in the open-field and square bridge tests. The authors reported that LA improved rotenone-induced behavioral deficits. In the open-field test, LA significantly increased the ambulation frequency, increased the number of stops, elevated the activity index and lessened the inactive sittings, but did not increase the rearing frequency in comparison to the group that received rotenone. In the Square bridge test, treatment with LA protected the rats from falling as compared to rotenone group (Zaitone et al., 2012).

Jalali-Nadoushan and Roghani (2013) investigated the effect of LA (at doses of 50 and 100 mg/kg) in a 6-hydroxydopamine (6-OHDA)-induced model of hemi-parkinsonism and observed significantly attenuated rotations on behavioral testing, induced by both doses.

The effects of LA in lipolysaccharide (LPS)-induced inflammatory PD model were also evaluated (Li et al., 2015), and the results showed that LA treatment (100 mg/kg/d) partially improved motor dysfunction. No significant recovery was observed in dyskinesia in PD mice that received LA. However, a significant amelioration was observed in the adhesive removal test, in which LA treatment significantly decreased the reaction time in comparison to the LPS group.

#### Huntington's Disease

Huntington's disease (HD) is a chronic neurodegenerative disease and a hereditary autosomal-dominant disorder of the central nervous system caused by a single genetic mutation (Ross et al., 2014), characterized by neuronal death in caudate and putamen and in the cerebral cortex, and to a lesser extent in hippocampus and subthalamic nucleus (Mehrotra et al., 2015). This disorder is classically characterized by motor symptoms and cognitive and behavioral features (Ross et al., 2014). A HD model that has been easily replicated in animals is based on the treatment with 3-nitropropionic acid (3-NP), which promotes development of mitochondrial dysfunctions leading to bioenergetic failure (energy impairment, oxidative stress, and excitotoxicity). A study by Mehrotra et al. (2015) investigated the effects of LA in 3-NP-induced HD in rats. Administration of LA improved spatial memory acquisition and retrieval assessed using the Morris water maze. Analysis of time taken and distance traveled to find the platform in the target quadrant revealed that LA supplementation for 21 days to 3-NP–treated animals resulted in a lower latency and the distance traveled was also reduced. In addition, the average number of platform crossings in the probe trial was increased in 3-NP treated animals that received LA. Thus, the authors demonstrated that LA supplementation improved spatial memory by ameliorating the iron- and copperinduced oxidative injury observed in age-related disorders. In the Y-maze test, animals display a preference to explore the novel arm of the maze, making fewer entrances in the previously explored arm, due to spontaneous alternation. The authors showed that 3-NP–treated animals traveled a significantly lower distance, assessed by the number of entries in the novel arm, and that supplementation with LA reversed these deficits.

#### Aging Models

Consistent evidence indicate that memory is affected by aging in rodents as well as in humans. A study by Liu et al. (2002a) investigated the effects of LA supplementation (0.1% in the diet) on spatial memory tested in the Morris water maze, and temporal memory using the peak procedure (time-discrimination procedure) in old rats. Results showed that LA supplementation alone or combined with another mitochondrial metabolite, acetyl-l-carnitine, improved both spatial and temporal memory. In 12-month old SAMP-8 mice, chronic LA administration improved cognition in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific locomotor effects (Farr et al., 2003).

LA has also been reported to improve behavior of aged mice in an open-field memory test (Stoll et al., 1993, 1994), in a Morris water maze test (Stoll et al., 1994; Liu et al., 2002a). LA also and ameliorated acquisition and retrieval in a dose-dependent manner, in old female NMRI mice, in the active avoidance learning test (Stoll et al., 1994).

#### Other Models of Neurotoxicity

Cui et al. (2006) evaluating a concomitant treatment with LA and d-Galactose exposure (used to induce memory loss and neurodegeneration) verified that LA ameliorated memory dysfunction in the Morris water maze task. Another study reported the neuroprotective effects of LA in neurotoxicity model induced by AlCl3 administration to mice (Mahboob et al., 2016). LA enhanced fear memory and social novelty preference in comparison to the AlCl3-treated group.

In summary, current evidence indicates that LA is able to improve memory, reversing impairments associated to a variety of experimental models of neurodegenerative disorders, and exposure to neurotoxicants, as well as normal aging. **Table 1** summarizes in vivo studies investigating the neuroprotective effects of LA on behavioral parameters. Furthermore, LA was also shown to act as a memory-improving molecule in different learning and memory paradigms, including aversive, spatial, and recognition memory.

LA has been tested in humans, in studies by Hager and coworkers (Hager et al., 2001, 2007), as a treatment option for AD. The authors examined the effect of LA for 24 and 48 months and observed that the treatment lead to a stabilization of cognitive function, verified by unchangeable records in two neuropsychological tests, mini-mental state examination (MMSE) and the AD assessment score, cognitive subscale (ADAScog).

### PUTATIVE MECHANISMS UNDERLYING LA-INDUCED NEUROPROTECTIVE EFFECTS

In vivo as well as in vitro studies have been performed in order to characterize cellular and molecular effects of LA underlying its memory-ameliorating activities (**Table 2**). The effects of LA on oxidative markers in various brain regions have been discussed in different studies in animals models of aging and neurodegenerative diseases (Cui et al., 2006; Ferreira et al., 2009; Militao et al., 2010; Farr et al., 2012).  Moreover, administration of LA reversed the augmentation of protein carbonyls levels in a radiation-induced cognitive dysfunction model (Manda et al., 2007), and decreased the protein carbonyls levels in aged SAMP8 mice (Farr et al., 2003).

A study by Zaitone et al. (2012) showed that LA increased striatal dopamine levels and significantly increased GSH and CAT activity in the striatum in a PD experimental model. In reserpine-treated rats, LA enhanced the amount of GSH, while diminishing GSSG levels in the striatum. Moreover, LA decreased NO concentrations in striatum and pre-frontal cortex, without significantly affecting S-nitrosothiol levels. LA also increased enzymatic activities of GPx and GST in the striatum (Bilska et al., 2007). Reserpine significantly decreased enzymatic activity of Lγ-glutamyl transpeptidase (γ-GT), while pretreatment with LA was able to restore it.

The effects of LA on oxidative stress in rotenone parkinsonian rat brains were investigated, showing that LA can reduce lipid peroxidation and protein carbonylation (Zaitone et al., 2012). LA also lowered the levels of MDA and nitrite in the 6-OHDA-induced rat model of hemi-parkinsonism (Jalali-Nadoushan and Roghani, 2013). Karunakaran et al. (2007) analyzed the protective effect of LA in the MPTP mouse model of PD, demonstrating that coadministration with LA prevents the activation of apoptosis signal regulating kinase (ASK1) signaling cascade and translocation of Daxx (death associated protein) in ventral midbrain and striatum, attenuating dopaminergic cell loss. R-LA induced significant reductions in markers of oxidative modifications in transgenic AD mice model, significantly decreasing HO-1 and protein-bound HNE levels (Siedlak et al., 2009). Inman et al. (2013) analyzed the effect of LA in the DBA/2J mouse model of glaucoma. The results showed that after 4 and 11 months of dietary LA, respectively, LA treatment increased antioxidant genes and protein expression, protected retinal ganglion cell (RGC), and improved retrograde transport. Dietary therapy also reduced lipid peroxidation, protein nitrosylation, and DNA oxidation in a retina model of glaucoma.

Accumulation of metal ions also has been associated with increased oxidative stress related with aging and neurodegenerative disorders. Suh et al. (2005) showed that LA supplementation can modulate age-related cortical iron accumulation, acting as metal chelator, thereby ameliorating age-associated oxidative stress. However, Liu et al. (2002b) showed that high concentrations of iron and copper found in old rats were not significantly decreased with LA supplementation.

There are multiple cell death mechanisms implicated in neurodegeneration. Apoptosis is a highly controlled cellular process that can be activated by two pathways: extrinsic, which is a receptor-mediated pathway, and intrinsic, which is mediated by signals from the mitochondria. Both pathways culminate at cleavage-dependent activation of aspartate-specific effector caspases (caspases-3, 6, and 7). Cui et al. (2006), using chronic systemic exposure of d-galactose in an aging model observed that a treatment with LA decreased caspase-3 protein levels and neuronal apoptosis, ameliorating neurodegeneration in the hippocampus. Manda et al. (2007), demonstrated that LA pretreatment protected against radiation. They observed that TABLE 1 | Summary of studies testing the effects of LA on behavioral parameters in animal models.

3-NP, 3-nitropropionic acid; AD, Alzheimer's disease; AlCl3, Aluminum chloride; HD, Huntington's disease; LPS, lipolysaccharide; 6-OHDA, 6-hydroxydopamine.

pre-treatment with LA prevented radiation-induced decreases of total, nonprotein and protein-bound sulfhydryl (T-SH, NP-SH, and PB-SH) levels in the cerebellum. Moreover, LA treatment also improved the cytoarchitecture of cerebellum, increasing the number of intact Purkinje cells and granular cells when compared to untreated irradiated mice.

Mehrotra et al. (2015) evaluated the effects of LA on mitochondrial dysfunctions in the 3-NP induced model of HD. The results showed that LA decreased malondialdehyde, protein carbonyls, reactive oxygen species and nitrite levels, and increased Mn-superoxide dismutase and CAT activity. They also found that LA improved histological and biochemical alterations, such as decreased cytosolic cytochrome c levels, caspase-3 and−9 activity and expression of apoptotic proteins (AIF, Bim, Bad, and Bax), suggesting its therapeutic efficacy in HD. LA improved activity of enzymes from the mitochondrial respiratory chain, altered cytochrome levels, increased histochemical staining of complex-II and IV, increased in-gel activity of complex-I to V, and increased mRNA expression of respiratory chain complexes.

Stoll et al. (1993) investigated the effect of LA on NMDA Receptor deficits in old female NMRI mice. The results showed that LA improved age-related NMDA receptor deficits (Bmax). No changes were observed regarding muscarinic, benzodiazepine, and α2-adrenergic receptor deficiencies. Thus, the authors concluded that LA-induced memory improving effects may be related to partial reparation of NMDA receptor deficits that accompany aging.

A loss of dopaminergic neurons is particularly relevant to PD, in which genetic and environmental factors are involved (Di Domenico et al., 2015; Li et al., 2015). Jalali-Nadoushana and Roghania using a rat model of hemi-parkinsonism (6-OHDA) found that LA prevented neuronal loss on the left side of the substantia nigra pars compacta (SNpc) (Jalali-Nadoushan and Roghani, 2013). In a study using the LPS-induced inflammatory PD model, Li et al. (2015) demonstrated that LA administration protected against dopaminergic neuron loss. Zaitone et al. (2012) observed that LA induced an increase in the number of neurons in the SNpc in rotenone parkinsonian rats. Li et al. (2015) reported that in addition to protecting against dopaminergic

#### TABLE 2 | Summary of in vivo studies testing cellular and molecular effects of LA.

 Moreover, the authors showed that LA inhibited the stimulation of nuclear factor-κB (NF-κB) and expression of pro-inflammatory molecules in M1 microglia. Zaitone et al. (2012) also investigated the effect of LA on mitochondrial DNA (mtDNA) integrity and quantity in the rotenone model of PD. The results showed that LA significantly decreased rotenone-induced mtDNA damage.

Liu et al. (2002a), examined the effects of LA on mitochondrial structure, and neurodegeneration in the hippocampus, and oxidative damage to nucleic acids in the hippocampus and cortex of aged rats. Dietary administration of LA significantly reduced oxidized RNA levels and reversed mitochondrial structural deterioration induced by aging in the hippocampus. Dwivedi et al. (2014) investigated the protective efficacy of LA against coexposure to arsenic-dichlorvos in rats. The results indicated that arsenic and dichlorvos induced oxidative stress and cholinergic dysfunction in brain, which was significantly protected by the supplementation with LA.

Seidman et al. (2000) indicated that age-associated mitochondrial impairment may be hampered by LA administration. Their results showed that mtDNA deletions associated with aging were reduced by LA and this effect appeared to be related to the mitochondrial capacity to protect and repair mtDNA against age-induced injury. Palaniappan and Dai (2007) investigated the effect of LA administration to aged rats and verified a reduction of mitochondrial lipid peroxidation, 8-oxo-dG and oxidized glutathione (GSSG) and increased GSH, ATP, and electron transport chain (ETC) complex activities in the brain.

The SAMP8 mouse strain is an experimental model that displays increased oxidative stress accompanied by memory decline associated to a rapid aging process. Proteomic analyses were used to examine differential protein expression and/or protein oxidative changes in brain samples from aged SAMP8 mice. In order to determine the mechanisms underlying LAinduced reversion of memory deficits exhibited by SAMP8 mice, Poon et al. (2005) analyzed the expression and specific carbonylation of proteins in brains from 12-month-old SAMP8 mice that received LA or vehicle. The levels of three proteins (neurofilament triplet L protein, a-enolase, and ubiquitous mitochondrial creatine kinase) were significantly increased, while protein carbonylation was reduced in lactate dehydrogenase B, dihydropyrimidinase-like protein 2, and a-enolase in aged SAMP8 mice that received LA, suggesting that, in addition to improving learning and memory, LA also can restore specific proteins in aged SAMP8 mouse brain.

Evidence indicates that deregulation in neurotransmitter systems, including decreased levels of neurotransmitters, decline in the number of receptors, and lower responsiveness to neurotransmitters can be key features of neurological disorders (Payton et al., 2005; Fidalgo et al., 2013). Arivazhagan and Panneerselvam (2002) investigated the effect of LA on levels of neurotransmitters (dopamine, serotonin, and norepinephrine), and showed that LA treatment can improve neurotransmitter function in models of neurodegenerative diseases. Jesudason et al. (2005) investigated the effect LA on levels of neurotransmitters in a model of AD by Aβ amyloid vaccination. The results showed that AD mice treated with LA exhibited enhanced levels of serotonin, dopamine, and norepinephrine, and the concentration of metabolites 5-hydroxyindole acetic acid (5- HIAA) and homovanillic acid (HVA) gradually returned to normal.

Ahmed (2012) explored the effect of LA on brain acetylcholinesterase (AChE) activity. The authors demonstrated that LA can ameliorate neurological injury related to Aβ and Al excess, by significantly restoring AChE activity. In addition, the authors showed that the treatment with LA restored the parameters of total homocysteine (tHcy), insulin, insulin like growth factor-1 (IGF-1), interlukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Mahboob et al. (2016), analyzed the effects of LA in AlCl3- model of neurodegeneration, demonstrating its capacity in ameliorating cognitive functions and enhancing cholinergic system functions. LA treatment increased the expression of muscarinic receptor genes M1, M2 and choline acetyltransferase (ChaT) relative to AlCl3-treated group.

There are many studies examining the neuroprotective actions of LA using in vitro models of neurodegeneration (Tirosh et al., 1999; Li et al., 2013; Xing et al., 2015), most of which focus on AD. For example, Ono et al. (2006) investigated the effects of LA and its reduced form, DHLA, on the formation, extension, and destabilization of β-amyloid fibrils (fAβ). The results showed that both LA and DHLA inhibited fAβ formation from amyloid β, as well as their expansion, and undermined preformed fAβs in a dose dependent manner. Lovell et al. (2003) also studied the effects of LA and DHLA in neuronal cultures challenged with amyloid β-peptide (Aβ 25-35), and observed that DHLA, but not LA, significantly protected against neurotoxicity induced by amyloid β-peptide and iron/hydrogen peroxide (Fe/H2O2).

In β-amyloid-intoxicated C6 glioma cells, LA increased cell viability and MnSOD expression. The increased GSSH and decreased GSH mitochondrial levels induced by Aβ were reversed by treatment with LA (Xing et al., 2015). In addition, LA protected cortical neurons against Aβ peptide- and hydrogen peroxide-induced damage, suggesting that the neuroprotective effects were partly related to PKB/Akt signaling pathway stimulation (Zhang et al., 2001).

The study by Deuther-Conrad et al. (2001) showed that the advanced glycation end products (AGE)-induced increases in oxidized glutathione were inhibited by R-LA in SH-SY5Y human neuroblastoma cells, indicating that AGE-mediated depletion of reduced glutathione follows the production of superoxide and hydrogen peroxide. de Arriba et al. (2003) investigated the effect of R-LA, in the same types of cells, on AGE accumulation, and found that AGE-induced metabolic changes were diminished by R-LA. Tirosh et al. (1999) showed that LA protected HT4 neuronal cells against glutamate-induced cytotoxicity, by inhibiting intracellular GSH depletion, and canceled the buildup of intracellular peroxide levels following the glutamate exposure.

Kamarudin et al. (2014) showed that R-LA ameliorated glutathione over glutathione disulfide ratio, decreased intracellular ROS levels and increased mitochondrial membrane potential in NG108-15 cells. In addition, R-LA stimulated the production of an anti-inflammatory cytokine, IL-10, inactivating glycogen synthase kinase-3b (GSK-3β) and decreasing both Bax/Bcl2 and Bax/Bcl-xL ratios. Suppression of NF-κβ p65 translocation and production of proinflammatory cytokines (IL-6 and TNF-α) followed inhibition of cleaved caspase-3. Yamada et al. (2011) investigated the effects of different isomers of LAs (racemate, R-LA, and S-LA) in human neuroblastoma SH-SY5Y cells. They showed that all types of LAs were effective in preventing cell death. R-LA and S-LA also enhanced expression of genes related to anti-oxidative response such as heme oxygenase-1 (HO-1) and phase II detoxification enzymes such as NAD(P)H:Quinone Oxidoreductase 1 (NQO1).

Other studies evaluated the effect of LA on in vitro model of PD. Li et al. (2013) showed that pretreatment with LA significantly prevented against apoptosis of PC12 cells elicited by MPP+, and inhibited intercellular ROS levels and mitochondrial transmembrane permeability, thereby protecting dopaminergic neuronal cells against oxidative damage. Moreover, Zhang et al. (2010) demonstrated that R-LA hindered rotenoneinduced mitochondrial dysfunction, oxidative damage, and α-synuclein and ubiquitin deposition in SK-N-MC human neuroblastoma cells.

# Involvement of LA in the Regulation of Cellular Signaling Pathways

LA has been proposed to exert a modulatory control on the cellular redox status. Due to its ability to be interconverted in one of its two forms—i.e., thiol, the reduced form and disulfide, the oxidized form—LA can regulate cellular redox environment by interacting with redox couples such as glutathione/glutathione disulfide, cysteine/cystine, and thioredoxin (Packer and Cadenas, 2011). LA has been described to regenerate other antioxidants, such as vitamin C and E, to increase GSH levels, and to provide modulation of proteins and transcription factors (Packer et al., 1995). Extracellular redox state is also regulated by LA, once its reduced form, DHLA, can interact with cystine, reducing it to cysteine, thereby stimulating its uptake by the cell, which in turn stimulates GSH synthesis (Han et al., 1997). Owing to the role played by LA in the regulation of thiol/disulfide redox couples, LA can be viewed as regulator of cell signaling and gene expression.

PI3K/Akt signaling pathway, critical to the regulation of cell growth, proliferation, differentiation, survival, and metabolism, has been shown to be modulated by LA. For instance, Jiang et al. (2013) have demonstrated that age-associated imbalance of PI3K/Akt was restored by LA treatment for 3 weeks in the drinking water in rats. LA treatment significantly increased Akt phosphorylation and lead to a recovery in the ratio pJNK/pAkt in cortex of aged rats. Prevention of sevoflurane-induced apoptosis by LA was accomplished through recovery of Akt and GSK3 β phosphorylation levels in the hippocampus (Ma et al., 2016). By activating PI3K/Akt signaling pathway, LA, administered for 3 days, was able to ameliorate cerebral ischemia and reperfusion-induced damage in adult rats (Dong et al., 2015). Sancheti et al. (2013) showed that LA increases brain glucose uptake and activates the insulin receptor substrate and the PI3K/Akt signaling pathway in a triple transgenic mouse model of AD (3xTg-AD), reversing the impaired synaptic plasticity and increasing input/output (I/O) and long-term potentiation (LTP).

# CONCLUDING REMARKS

Compelling evidence indicates that LA displays memoryameliorating properties in a variety of experimental models of neurodegenerative diseases, as well as in memory decline associated with aging in rodents. Studies aiming to assess the neuroprotective effects of LA on behavioral outcomes showed that LA can reduce memory deficits in different behavioral paradigms on AD (Quinn et al., 2007; Farr et al., 2012), HD (Mehrotra et al., 2015), oxidative stress (Stoll et al., 1993, 1994; Liu et al., 2002a; Farr et al., 2003; Manda et al., 2007), and age-associated cognitive dysfunction (Cui et al., 2006; Mahboob et al., 2016) models. In humans, two studies in AD patients have supported the positive cognitive effects of LA (Hager et al., 2001, 2007).

Many studies reported beneficial effects of LA in the rat brain or neuronal cell cultures, using different molecular markers of oxidative stress, such as reduction in the levels of lipid peroxides and protein carbonyls, recycling endogenous antioxidants such as vitamin C and E, increasing glutathione levels (Packer et al., 1997; Di Domenico et al., 2015; Mehrotra et al., 2015), inhibiting free radical formation, chelating transition metal ions such as iron, thus reducing its bioaccumulation in the brain (Moini et al., 2002; Shay et al., 2009; Rochette et al., 2013). LA was also shown to display anti-inflammatory properties (Deuther-Conrad et al., 2001; Li et al., 2015), and affect cell death. In vivo and in vitro studies showed that LA ameliorated neurodegeneration in the hippocampus, decreasing neuronal apoptosis and caspase-3 protein levels, supporting a neuroprotective role mediated by the mitochondrial cell death pathway. These effects suggest that LA is able to improve mitochondrial dysfunctions. Interestingly, in addition to decreasing neuronal cell death, LA also inhibited fAβ formation from amyloid β-protein, ameliorating the neurological damage induced by Aβ, and significantly restored AChE activity. This evidence suggests that LA presents a potential role in enhancing cholinergic and cognitive functions. These neuroprotective effects may be related to the properties of LA in ameliorating memory loss associated to neurodegenerative diseases.

Remarkably, LA was able to reverse age-associated glutamatergic NMDA receptor deficits (Stoll et al., 1993), which might be centrally related to LA memory-improving effects. LA was also shown to improve the function of neurotransmitter systems, including dopamine, serotonin, and norepinephrine. Taken together, these findings provide evidence that LA can reverse loss of neurotransmitters, their receptors and responsiveness to neurotransmitters, which can underlie its effects on cognitive functions.

In summary, this review has described and discussed relevant studies investigating the effects of LA on cognition as well as its cellular and molecular effects, aiming to improve the understanding of the therapeutic potential of LA in memory loss during aging and patients suffering from neurodegenerative disorders. Although the mechanisms of action of LA are not fully understood, multiple pathways are likely to be involved in its neuroprotective properties. The memory-improving effects and neuroprotective actions of LA support its use as an adjuvant treatment for neurodegenerative disorders.

# AUTHOR CONTRIBUTIONS

PM has performed literature search and has written the first draft of the manuscript. NS has extensively revised and contributed in writing the final version of the manuscript.

#### REFERENCES

# FUNDING

This research was supported by the National Council for Scientific and Technological Development (CNPq; grant number 308290/2015-1 to NS).

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stress in a mouse model of Alzheimer's disease. Free Radic. Res. 43, 156–164. doi: 10.1080/10715760802644694

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Molz and Schröder. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# β-Sheet Breaker Peptide-HPYD for the Treatment of Alzheimer's Disease: Primary Studies on Behavioral Test and Transcriptional Profiling

Weiying Liu1 \* † , Fengxian Sun2†, Moxin Wan2 , Fang Jiang2 , Xiangyu Bo3 , Laixiang Lin4 , Hua Tang1 and Shumei Xu2 \*

#### Edited by:

*Antonella Gasbarri, University of L'Aquila, Italy*

#### Reviewed by:

*Maria Grazia Morgese, University of Foggia, Italy Luigia Trabace, University of Foggia, Italy*

#### \*Correspondence:

*Weiying Liu<EMAIL_ADDRESS>Shumei Xu<EMAIL_ADDRESS>† These authors have contributed equally to this work.*

#### Specialty section:

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology*

Received: *21 September 2017* Accepted: *19 December 2017* Published: *08 January 2018*

#### Citation:

*Liu W, Sun F, Wan M, Jiang F, Bo X, Lin L, Tang H and Xu S (2018)* β*-Sheet Breaker Peptide-HPYD for the Treatment of Alzheimer's Disease: Primary Studies on Behavioral Test and Transcriptional Profiling. Front. Pharmacol. 8:969. doi: 10.3389/fphar.2017.00969* *1 Department of Pathogen Biology, Tianjin Life Science Research Center, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China, 2 Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China, 3 Department of Pathology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China, 4 Key Laboratory of Hormone and Development (Ministry of Health), 2011 Collaborative Innovation Center of Tianjin for Medical Epigenetics, Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, China*

Background: Alzheimer's disease (AD), is a progressive neurodegenerative disease that is characterized by cognitive loss. Most researchers believe that aggregation and accumulation of β-amyloid peptides (Aβ) in brain cells are the central pathological hallmark of this disease.

Methods: Based on the amyloid hypothesis, a 10 amino acids β-sheet breaker peptide HPYD (His-Lys-Gln-Leu-Pro-Phe-Tyr-Glu-Glu-Asp) was designed according to the structure and sequence of the previous designed peptide H102. Accelerated stability test, thioflavine T (ThT) fluorescence spectral analysis and transmission electron microscopy (TEM) imaging were performed to detect the stability and inhibitory effects on the aggregation of Aβ1−42 by H102 and HPYD. FITC-labeled HPYD was first tested to determine whether it could be transferred along the olfactory pathway to the brain after nasal administration to mice. Subsequently, the Morris Water Maze (MWM) test for behavioral analysis was used to investigate the learning and memory ability of APP/PS1 transgenic mice by HPYD. Immunohistochemistry and western blot analysis was performed to determine the role of HPYD on Aβ and APP protein levels. In addition, microarray analysis was used to evaluate the effect of HPYD on gene expression in AD mouse models.

Results: Our *in vitro* results demonstrated that HPYD had enhanced stability and inhibitory effects on Aβ1−42 aggregation compared to H102. HPYD could be delivered into the brain through nasal administration and improved the learning and memory ability in APP/PS1 transgenic mouse models by reducing Aβ and APP protein levels. In addition, microarray analyses suggested that several genes related to the inflammatory pathway,

**346**

AD and gluco-lipid metabolism were dysregulated and could be restored to almost normal levels after HPYD administration to mice.

Conclusions: Our results demonstrated that HPYD could be a potential therapeutic drug candidate for the treatment of AD.

Keywords: Alzheimer's disease, HPYD, β-amyloid peptides, APP, β-sheet breaker peptide

# INTRODUCTION

Alzheimer's disease (AD) is a complex, severe neurodegenerative disorder of the central nervous system, which manifest as progressive cognitive decline and memory impairment in the elderly and presenium individuals. More than 44 million people are currently living with AD and the global health care cost in 2010 was about US\$818 billion (Weiner et al., 2013). The incidence of AD has been increasing every year, and by 2050 about 131.5 million people will suffer from dementia if there are no effective therapies (Cummings et al., 2016). Although AD has led to a major social and health care problem, there is still no absolute effective treatment for the disease. Hence, it is critical to develop novel drugs and effective therapies for AD.

The pathological characteristics of AD mainly include massive senile plaque deposits, neurofibrillary tangles as well as selective loss of neurons and synapses in specific brain regions, such as the cerebral cortex and hippocampus (Mattson, 2004; Shen and Kelleher III, 2007). Senile plaques contain extracellular deposits of β-amyloid peptide in its fibrillar form and neurofibrillary tangles, which are composed of hyper phosphorylated tau protein. There are many hypothesis regarding the pathogenesis of AD, such as the cholinergic hypothesis (Francis et al., 1999), amyloid hypothesis (Hardy and Allsop, 1991; Mudher and Lovestone, 2002), tau hypothesis (Goedert et al., 1991; Mudher and Lovestone, 2002) and several other hypotheses (Reisberg et al., 1999; Deane and Zlokovic, 2007). The amyloid hypothesis has become the most accepted mechanistic hypothesis for AD pathogenesis, although some amendments have been made.

β-amyloid peptides (Aβ) are 39–42 amino acid peptide residues that are derived from putative intramembranous processing of amyloid precursor protein (APP) by γsecretase/PS1 aspartyl protease (Selkoe, 1999; Hardy and Selkoe, 2002). Only a small amount of Aβ is generated by the cleavage of APP by β- or γ-secretase in healthy individuals, while a large amount of APP is metabolized by α- and β-secretase without Aβ generation (Haass et al., 1992; Zhang and Xu, 2007). However, genetic mutations in β- or γ-secretase in AD patients results in increased APP cleavage activity, which subsequently generates large amounts Aβ. Aβ has been shown to aggregate and accumulate abnormally in the brain of AD patients, and extracellular amyloid plaques of Aβ peptides aggregation can trigger a cascade of pathologic events leading to nerve fiber entanglement and neuronal apoptosis (Hardy and Selkoe, 2002; Karran et al., 2011).

Recently, it has been demonstrated that smaller and more soluble aggregates, including a variety of compounds of soluble oligomers, ADDLs (amyloid β-derived diffusible ligands) or protofibrils to be the predominant toxic forms of Aβ (Walsh and Selkoe, 2007; Allsop and Mayes, 2014; Karran and De Strooper, 2016; Selkoe and Hardy, 2016). Compared to fibrillar plaques, Aβ oligomers (AβOs) are considered to be the main mediators of cytotoxicity in AD (Ashe and Aguzzi, 2013). The oligomeric Aβ can initiate the phosphorylation of Src kinase Fyn, asparagine endopeptidase (AEP) and GSK3-β, which then can subsequently induce the hyperphosphorylation of tau protein (Martin et al., 2013; Zhang et al., 2014). AβOs and ADDLs bind to synaptic contacts and cellular membranes more rapidly and with higher affinity than fibrillar Aβ (Aβf). This binding compromises the integrity of intracellular membranes to induce an elevation of intracellular Ca2+, which then results in rapid and massive neuronal cell death (Demuro et al., 2005; Deshpande et al., 2006). In addition, Aβ also could upregulate inflammatory cytokines and increase the nitric oxide release to cause neuroinflammation (Hu et al., 1998). Several studies have demonstrated that Aβ can induce the expression of several inflammatory factors, including CASP1, CASP4, PLA2G4A, and PTPRC (Lee et al., 2011; Zhu et al., 2011; Mehta et al., 2012; Kajiwara et al., 2016). It has been reported that soluble Aβ1−42 protofibrils could stimulate microglial production of tumornecrosis factor α (TNFα) to stimulate inflammatory responses (Paranjape et al., 2013). Taking all these observations and studies into consideration, the amyloid hypothesis is recognized as the most prominent theory to explain the pathogenesis of AD.

It is thought that Aβ aggregations are generated due to Aβ clearance deficiencies of γ-secretase (Jarrett et al., 1993; Golde et al., 2000; McGowan et al., 2005). Hence, many secretase inhibitors were developed as the initial small-molecular therapies for AD (De Strooper et al., 2010). Semagacestat, a γ-secretase inhibitor, reached Phase 3 clinical trials, but was halted due to adverse events of worsening cognition and activity, as well as increasing the incidence of skin cancer (Karran and De Strooper, 2016). Many other strategies were also considered for AD, for example, inhibiting Aβ aggregation. Tramiprosate was found to inhibit Aβ aggregation by maintaining Aβ in a non-fibrillar form, thus inhibiting amyloid deposition (Gervais et al., 2007). Tramiprosate could significantly reduce brain amyloid plaque load (∼30%) and the cerebral levels of soluble and insoluble Aβ40 and Aβ42 (∼20–30%) in TgCRND8 mice (Gervais et al., 2007). However, no significant therapeutic effects were observed as primary outcomes in the Alzheimer Disease Assessment Scale-cognitive subscale (ADAS-cog) and Clinical Dementia Rating-Sum of Boxes (CDR-Sum of Boxes) in phase III trials (Aisen et al., 2011). Nonetheless, ALZ-801, a novel prodrug of tramiprosate, has shown excellent oral safety and tolerability, and its PK characteristics were significantly improved compared to oral tramiprosate in phase I studies (Hey et al., 2017). Additionally, immunotherapies were considered

for AD therapy. Bapineuzumab and solanezumab represent two humanized monoclonal antibodies that increases the clearance of Aβ by specifically targeting amino acids 1–5 and 16–24 of Aβ peptide, respectively. However, Phase 3 clinical trials of bapineuzumab were halted after the completion of two trials because it did not improve clinical outcomes in patients with AD (Salloway et al., 2014). Similarly, solanezumab also failed to meet the primary outcome in Phase 3 clinical trials (Siemers et al., 2016). Although many amyloidocentric drugs have failed after Phase 3 clinical trials, it does not diminish the pathogenic theory of this disease.

Aβ consists of a hydrophobic carboxyl terminus and a hydrophilic amino terminus. The hydrophobic carboxyl terminus of Aβ mainly consists of β-sheets while the hydrophilic amino terminus mainly consists of α-helix and β-turns (Chou and Fasman, 1977). Aggregation of monomeric Aβ into oligomers are formed through the internalization of the hydrophobic carboxyl terminus and exposing the hydrophilic amino terminus (Hilbich et al., 1992). The carboxy terminus of Aβ is critical for amyloid formation.

β-sheet breaker peptides (also referred to as peptidic inhibitors) are a class of compounds that are highly potent in ameliorating Aβ1−42- or α-synuclein-inflicted cell toxicity (Watanabe et al., 2002; El-Agnaf et al., 2004). β-sheet breaker peptides are homologous to regions of the β-sheet hydrophobic carboxyl segments and highly effective in inhibiting Aβ amyloidogenesis (Jarrett et al., 1993). Based on the amino acid residues 17–21 of Aβ1−42, we previously designed a β-sheet breaker peptide H102 (His-Lys-Gln-Leu-Pro-Phe-Phe-Glu-Glu-Asp) that can reduce amyloid load and cerebral damage and improve the learning and memory ability of AD animal models (He et al., 2008; Lin et al., 2014). However, H102 was not very stable. Hence, we designed an alternative β-sheet breaker peptide HPYD (His-Lys-Gln-Leu-Pro-Phe-Tyr-Glu-Glu-Asp) by substituting Phe with Tyr. The stability of HPYD and the ability to inhibit Aβ aggregation were studied in vitro. HPYD displayed excellent stability and the ability to inhibit Aβ aggregation compared to H102. However, HPYD efficacy for the treatment of AD in vivo remained to be elucidated.

In this study, we first compared the stability of H102 and HPYD using the accelerated stability test, and then performed inhibitory studies on the aggregation of Aβ1−42. We also investigated the ability of HPYD to transverse into the brain through the olfactory pathway after nasal administration of fluorescein isothiocyanate (FITC)-labeled HPYD. The effect of HPYD on APP/PS1 transgenic mice behavior and the APP and Aβ expression in the brain were also investigated. Furthermore, we profiled the gene expression in normal mice (control group), APP/PS1 transgenic mice (model mice) and APP/PS1 transgenic mice treated with HPYD (HPYD group) using gene microarrays. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to annotate their functions. Our findings not only has important implications for the potential treatment of AD using HPYD, but also provide insights to the mechanism of Aβ toxicity in AD patients and for the development of new therapeutic strategies for AD.

# MATERIALS AND METHODS

#### Animals

Nude mice (weight: 26 to 28 g; age: 2 months), APP/PS1 mice (weight: 25.3 to 28.1 g; age: 8 months) and C57/6J mice (weight: 25.5 to 28.6 g) were purchased from the institute of laboratory animal sciences, CAMS & PUMC (Chinese Academy of Medical Sciences and Peking Union Medical College). APP/PS1 mice have been previously demonstrated to form amyloid plaques, which have been approved by institute of laboratory animal sciences, CAMS and PUMC. All animal experiments were performed in accordance with the China Physiological Society "Guiding Principles in the Care and Use of Animals" approved by Tianjin Medical University Animal Care and Use Committee (NO. 20130021).

#### Compounds

H102, FITC-labeled HPYD and HPYD were synthesized using the Fmoc solid-phase synthesis method and purified by HPLC (Gill Biotechnology Company, Shanghai, China). The compounds were greater than 95% pure as measured by HPLC-MS. HPYD, a polypeptide comprising the amino acid (AA) sequence of His-Lys-Gln-Leu-Pro-Phe-Tyr-Glu-Glu-Asp, was dissolved in normal saline.

#### Accelerated Stability Test

0.5 mg of HPYD or H102 was dissolved in 1 mL of 0.2 mol/L phosphate buffer containing 100 mg/L of trypsin. The trypsin solution and polypeptide were mixed at a ratio of 1:4, and the solution was placed in a temperature controlled water bath shaker at 37◦C for 0, 40, 100, 160, 220, 280, 340, and 400 min. The samples were then heated to 80◦C for 10 min, and the stability of HPYD and H102 was detected at the different time points. High performance liquid chromatography (HPLC) analysis of HPYD and H102 was performed using a Phenomerex C18 column (250 mm × 4.6µm, 5µm; Sigma, Inc. U.S.A.). The mobile phase consisted of solvent A, 0.1% TFA in acetonitrile and solvent B, 0.1% TFA in water at a ratio of 22.5:77.5 (v/v). The sample injection volume was 20 µL. The flow rate was 1 mL/min, and the detection wavelength was 220 nm.

## Thioflavine T (ThT) Fluorescence Spectral Analysis

Aβ1−42 freeze-dried powder was dissolved in 50 mmol/L sodium phosphate buffer solution (pH = 7.4) to a concentration of 22.15 µmol/L, and H102 and HPYD were dissolved in the same PBS solution to a concentration of 88.60 µmol/L. Aβ1−42 solution was then mixed with H102 and HPYD respectively in equal volumes. The Aβ fibrils were grown at 37◦C for 24 hrs. 10 µL solution from each group was then added to 990 µL of 3.0 µmol/L ThT solution and fluorescent intensity was measured using a VARIAN PTC-Au00-01058 fluorescence spectrophotometer (VARIAN, USA) with an excitation wavelength of 453 nm and emission of 478– 486 nm.

The Aβ1−42 samples (11.07 µmol/L 20 µL) were incubated in 37◦C for 5 days, and the mixture of Aβ1−42 (22.15 µmol/L 10 µL) with HPYD (88.61 µmol/L 10 µL) or H102 (88.61 µmol/L 10 µL) were incubated under the same conditions for 5 days. Aliquots (5 µL) of each sample was spotted onto thin carbon substrates supported by carbon film on a 300 mesh copper grid for 15 min and blotted dried. The TEM grids were negatively stained with 2% uranyl acetate for 2 min and air dried. The samples were subsequently imaged using a Hitachi H-600 transmission electron microscope.

## Fluorescence Imaging System Analysis

Nude mice were anesthetized using 20% urethane (5 mL/kg), and then placed on the VFIS observation platform. Nude mice were then irradiated at 490 nm excitation without treatment, and then irradiated for 5, 15, and 30 min after nasal administration with FITC-HPYD (5.535 mg/kg). Thirty minutes after nasal administration with FITC-HPYD, the mice were euthanized and the hippocampus, cortex, olfactory bulb, heart, lung, liver, spleen and kidneys were removed. These organs were then irradiated at 490 nm excitation.

### HPYD Treatment

The APP/PS1 transgenic mice were randomized into the model and HPYD treatment group (n = 12 for each group). C57BL/6J mice with the same genetic background and age served as the normal controls. Treatment group received intranasal administration of HPYD saline solution (33 mg/mL, 5 µL/d), while the normal control group and model group were given with the same volume of saline for 30 days.

## Behavioral Test

The behavioral test was performed after 30 days of intranasal administration using the Morris Water Maze (MWM) according to our previous report (Lin et al., 2014). Briefly, the MWM test was conducted in a circular pool with a diameter of 80 and 32 cm deep. The pool was filled with 25 ± 1 ◦C water to a depth of 1 cm higher than the escape platform. The water was made opaque white to hide the escape platform. The orientation and navigation experiments were conducted for 5 days to record the time intervals when the animals climbed onto the platform. If animal failed to find the hidden platform within 90 s, the mouse was placed back onto the platform for 20 s, and the escape latency was recorded as 90 s. The platform was placed in the center of the third quadrant and remained in the same position throughout the orientation and navigation experiment. On the 6th day, the platform was removed, and each mouse was allowed to swim freely for 90 s to record the frequency of passing the hidden platform and the original angle.

### Immunohistochemistry

At the end of the MWM test, the mice were euthanized and their brains were removed and rapidly placed on ice. The brain tissues were then fixed in 4% paraformaldehyde solution and waxed. Tissue sections were then dewaxed, and antigen retrieval was performed using boiling citrate buffer solution. This was followed by incubating the sections in 3% H2O2 solution at room temperature for 10 min to block endogenous peroxidase activity. The sections were then blocked by incubating with normal goat serum at room temperature for 15 min, and subsequently diluted antibodies (Aβ: 1:100; APP: 1:100) were added and incubated overnight at 4◦C. Biotin-labeled secondary antibody was added and incubated for 15 min at room temperature, followed by incubation with strept avidin-biotin complex (SABC) and DAB chromogenic reagent. Finally, the sections were counterstained with hematoxylin and observed under a microscope.

## Western Blotting

Western blot analysis was performed according to the detail procedures described previously. Primary Aβ antibody (1:200) was purchased from Abcam (USA) and anti-APP antibody (1:200) was purchased from Wuhan BOSTER Bio Company (Wuhan, China). The secondary goat anti-rabbit antibody was purchased from Sigma-Aldrich (St Louis, MO, USA).

# Microarray Analysis

Microarray hybridization was carried out by Shanghai GMINIX Biotech Limited Company (China) using the GeneChip R Mouse Gene 1.0 ST Array (Affymetrix, USA Scientific) with 770,317 probes. Briefly, total RNA was extracted from frozen brain tissues using the RNeasy mini kit (Qiagen, Valencia, CA) and used for cDNA synthesis using the cDNA synthesis kit (Affymetrix, Inc., USA). cDNA was labeled using the Gene Chip2 WT Terminal Labeling Kit (Affymetrix, Inc., USA), and then the labeled cDNA was hybridized to the mouse Gene chip at 45◦C for 16 h. The Gene chip was then washed with wash solution A, wash solution B and deionized water, and stained using Cocktail 1 and Cocktail 2. The Gene Chip 2 Scanner 300 7G (Affymetrix Inc.) and the AGCC Scan Control software was used for date analysis. The gene ontology (GO) enrichment analysis was performed using the GOEAST software toolkit (P ≤ 0.05), and signaling pathway analysis was performed using the KEGG data software. Software Matlab 7.1 and java, was used to build and analyze the dynamic Gene networks, and network maps of the differentially expressed genes were constructed from the different states.

### Accession Numbers

The Gene Expression Omnibus accession number for normal mice, model mice and HPYD mouse expression profiles is GSE104249.

# Real-Time Quantitative PCR (RT-qPCR)

RT-qPCR analysis was performed using an ABI 7500 thermocycler (Applied Biosystems) with UltraSYBR Mixture purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China). Gene transcript normalization was performed using housekeeping gene β-actin. All primers used for RT and qPCR are listed in **Table 1**.

### Statistical Analysis

All experiments were performed at least three times. Data is presented as mean ± SD. The date of escape latency from MWM were analyzed by multivariate analysis of variance (ANOVA) and the other data were analyzed by one-way ANOVA, followed by

#### TABLE 1 | Primers used in this study.

Student-Newman-Keuls test. All the analysis was performed by SPSS statistical software (version 21, IBM, Armonk, NY). P ≤ 0.05 was considered to be statistically significant (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

### RESULTS

# HPYD had a Better in Vitro Stability and Inhibitory Effect on Aggregation of Aβ1−42 Compared to H102

To investigate the stability of HPYD in vitro, accelerated stability test using trypsin was performed. After treatment with 100 mg/L trypsin at 37◦C for 400 min, followed by heat treatment for 10 min at 80◦C, the concentration of HPYD was 81.67% ± 0.42, while the concentration of H102 was only 41.07% ± 0.17, indicating that the stability of HPYD was better compared to H102 (**Table 2**). In addition, to compare the inhibitory effects on aggregation of Aβ1−42 with H102, ThT fluorescence spectral analysis and TEM was performed. Results showed that HPYD could significantly inhibit the aggregation of Aβ42 compared to H102 (**Figures 1A,B**). Taken together, this demonstrates that HPYD had better in vitro stability and inhibitory effects on Aβ1−42 aggregation compared to H102.

# Uptake of FITC-HPYD Into the Brain after Nasal Administration

HPYD, a β-sheet breaker peptide of 10 AAs, is difficult to administer orally or intravenous because it easily undergoes rapid in vivo degradation in the gastrointestinal tract and blood. Therefore, to determine whether HPYD can be administered to the brain through the olfactory pathway after nasal administration, HPYD was labeled with FITC and observed by fluorescence imagining of the brain. Before nasal administration, nude mice were irradiated at 490 nm excitation wavelength, and then were subsequently administrated with FITC-HPYD though nasal cavity and irradiated after 5, 15 and 30 min with wavelength of 490 nm. Results showed that FITC-HPYD first appeared at the brain area at 5 min after nasal administration, and the fluorescence gradually expanded with time. As shown in **Figure 2A**, 30 min after nasal administration, the fluorescence had spread over the whole body of the nude mouse, however, the fluorescence intensity in the brain was the strongest compared to other organs. The mice were then euthanized and the hippocampus, cortex, olfactory bulb, heart, lungs, liver, spleen and kidneys were removed at 30 min after nasal administration and irradiation. The results indicated that the fluorescence intensity of the olfactory bulb was the strongest, with the cortex and the hippocampus having the second and third strongest intensities, respectively. The other major organs showed varying degrees of fluorescence intensity. Among these organs, the lungs had the strongest intensity, followed by the liver (**Figure 2B**). These results demonstrated that FITC-HPYD could be delivered into the brain through nasal administration.

# HPYD Improves the Learning and Memory Ability in APP/PS1 Transgenic Mice

MWM test for behavioral analysis is a common strategy to investigate the learning and memory ability in AD mouse models. To determine the efficacy of HPYD on APP/PS1 transgenic mice, the MWM test was conducted for 6 days. First, the escape latency test was performed from Day 1 to Day 5. As shown in **Figures 3A,B**, the control group and the HPYD group displayed no significant differences from Day 1 to Day 5. In addition, the control group vs. the model group, or the model group vs. the HPYD group, showed no significant difference from Day 1 to the Day 3. However, significant differences were observed between the HPYD group and the model group at Day 4, and in addition, statistical significant differences between the model group and control group were observed on Day 4. The

*aPercentage of initial concentration (Mean* ±*\_SD [%]; n* = *3) of H102 (400 mg/L) and HPYD (400 mg/L) remaining after treatment with 100 mg/L trypsin at 37*◦*C for 400 min, followed by heat treatment for 10 min at 80*◦*C.*

escape latency test indicated that the APP/PS1 transgenic mice, normal mice and the HPYD group mice all required time to find the platform at the start of the test, however, the learning and memory ability demonstrated significant differences after treatment with HPYD in APP/PS1 transgenic mice after 3 days of training. On the 6 day, spatial probe test was performed on the mice without the platform. As shown in **Figures 3C–E**, memory ability was estimated by the frequency passed the hidden platform (**Figure 3C**) and original angle (**Figure 3D**). Frequency passed the hidden platform in the target quadrant displayed significant differences between the model group and the control group, indicating that the model group had a poor ability to find the platform compared to the control group. However, the HPYD group demonstrated an improved ability to find the platform compared to the model group. No significant differences were observed between the HPYD group and the control group. Finally, the ability to locate the original angle was also investigated at the 6th day. The HPYD group was able to find the location of the original angle much quicker compared to the model group, indicating that the HPYD group mice had relatively good memory. No significant differences were observed between the control group and the HPYD group. Collectively, these results indicate that HPYD could improve the learning and memory ability in APP/PS1 transgenic mice after nasal administration of HPYD.

#### HPYD Reduces Aβ and APP Protein Levels in APP/PS1 Transgenic Mice

HPYD is a novel β-sheet breaker peptide that could inhibit Aβ1−42 aggregation in vitro. To determine the effect of HPYD on Aβ in vivo, HPYD was administrated though the nasal cavity into the brain. Tissues were then harvested after 6 days for immunohistochemistry and western blot analysis. As shown in **Figure 4A**, the hippocampus CA1 and cortex had higher expression of Aβ in the model group mice compared to control group mice, while Aβ was lower in the HPYD group compared to the model group. No significant differences were observed between the control group and the HPYD group. In addition,

western blot analysis showed that Aβ was highly expressed in the hippocampus and cortex of the model group mice compared with the control group, while Aβ was expressed at a lower level in the HPYD group mice compared to the model group mice (**Figure 4B**). There were no statistical significant differences between the HPYD group mice and control group mice. APP, the precursor of Aβ, is upregulated in the brain tissues of AD patients. To determine whether HPYD had an effect on APP expression, we determined the expression of APP using immunohistochemistry and western blot (**Figures 4B,C**). Results showed that APP was upregulated in the brain tissues of the model group mice compared to the control group mice, while it was downregulated in the HPYD group mice compared with the model group mice. The expression of APP in the HPYD group mice was similar to the control group. These results indicate that HPYD can reduce Aβ and APP protein levels in APP/PS1 transgenic mice.

# Transcriptional Profiling of Brain Tissues from APP/PS1 Transgenic Mice after Treatment with HPYD

To investigate the effect of HPYD on gene expression of APP/PS1 transgenic mice, the brain tissues of the control group, model group and HPYD group mice were harvested at the termination of the MWM test. Total RNA from brain tissues were isolated and microarray analysis was performed by Shanghai GMINIX Biotech Limited. Based on microarray analysis, 119 genes with significant differences were selected for further analysis (**Figure 5A**). Among the selected genes, 15 genes were markedly down-regulated in the model group compared to the control group, however, the expression levels of these genes were restored to normal levels after treatment with HPYD. Additionally, the majority of the selected genes were up-regulated in the model group compared to the control group, which were restored to normal levels after treatment with HPYD. These differentially expressed genes were then analyzed

by GO analysis to predict their function. The most enriched GO terms are shown in **Figure 5B**, and includes; protein binding, extracellular exosome, immune system, inflammatory reaction and gluco-lipid metabolism. KEGG pathway analysis showed that these genes were involved in many inflammatory response pathways, including viral carcinogenesis, RIG-I-like receptor signaling and NOD-like receptor (NLR) signaling pathway (**Figures 5C,D**). Co-expression network analysis revealed gene-function relationships of the differential gene expressions, including CDK1, PLA2G4A, APP and SCN9A, which were reported to be involved in AD (Ling et al., 2003; Schaeffer et al., 2010; Hilgeroth et al., 2014). In addition, many

inflammatory related genes, such as CLEC4D and CLEC5A were differentially expressed. These two genes are members of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily that play important roles in inflammation and immune response (Wu et al., 2013; Wilson et al., 2015). Other genes of interest include; Inflammasome-related CASP1 (encodes caspase-1) and CASP4 (encodes caspase-4) that mediates non-canonical activation of the NLRP3 inflammasome (Schmid-Burgk et al., 2015). NAIP2 and NAIP5, belong to the NAIP/NLRC4 inflammasomes, and play important physiological roles in antibacterial defense and inflammation (Diebolder et al., 2015; Zhang et al., 2015). In addition, 18 genes associated with AD or inflammatory responses were selected for validation by RT-qPCR. There was a good concordance in the expression levels of these genes by RT-qPCR and microarray (**Figure 6**). Taken together, the above results indicated that dysregulated expression of several genes in the model group mice could be restored to normal levels after treatment with HPYD, indicating that HPYD may be a potential therapeutic candidate for treatment of AD.

### DISCUSSION

Alzheimer's disease is the most common dementing disease worldwide. Over the past decade, several potential therapeutics were evaluated in clinical trials, but only five drugs (Memantine and four cholinesterase inhibitors) have been approved worldwide to treat AD (Cummings et al., 2016). However, due to the modest clinical efficacy on temporarily ameliorating memory and thought, these five drugs are not very efficacious in treating the underlying cause of AD and preventing the rate of cognitive

decline. Therefore, it is critical to develop novel drugs for the treatment of AD.

The amyloid hypothesis is one of the most accepted mechanistic hypothesis for AD posits that dysregulated aggregation and accumulation of Aβ in the brain leads to nerve fiber entanglement and neuronal apoptosis (Hardy and Selkoe, 2002). The amyloid hypothesis implies that decreasing the levels of Aβ or inhibiting Aβ aggregation could lead to a cure for AD. Based on this hypothesis, many drugs were developed for the treatment of AD. Tramiprosate, a drug of potential

interest for the treatment of AD, inhibits Aβ aggregation and amyloid deposition (Gervais et al., 2007). Bapineuzumab and solanezumab, two humanized monoclonal antibodies that specifically target amino acids 1–5 or 16–24 of Aβ peptide, increases the clearance of Aβ and decreases amyloid deposition in the brain (Salloway et al., 2014). Despite efficacy in AD animal models, the above mentioned drugs all failed in Phase 3 clinical trials due to efficacy. However, the failure of several amyloidocentric drugs for the treatment of AD did not diminish the pathogenic theory of this disease. Numerous novel drugs have been designed or screened for inhibiting Aβ production. For example, Icariside II (ICS II), an anti-cancer natural compound extracted from Herba Epimedii Maxim, was demonstrated to inhibit Aβ production by reducing APP and BACE1 expression in APP/PS1 transgenic mice (Yan et al., 2017). ALZ-801, a novel prodrug of tramiprosate, has been shown to have excellent oral safety and tolerability compared to tramiprosate though phase I studies (Hey et al., 2017).

Previously, we designed a β-sheet breaker peptide, H102, that could inhibit Aβ aggregation as well as reducing amyloid load in the brains of APP/PS1 transgenic mice, thus limiting brain damage and improving the symptoms of AD in animal models (He et al., 2008; Lin et al., 2014). However, H102 was not stable in vitro. We designed alternative β-sheet breaker peptide, HPYD, by substituting Phe7 with Tyr7. This peptide showed higher in vitro stability and better efficacy of inhibiting Aβ aggregation compared to H102. In addition, FITC-labeled HPYD demonstrated that the peptide could enter the brain after nasal administration. MWM test demonstrated significant spatial learning and memory disorders in APP/PS1 transgenic model mice, which were in concordance with previous studies (Yan et al., 2017). Several studies have also demonstrated that Aβ and APP protein expression levels were markedly increased in APP/PS1 transgenic model mice. These outcomes were effectively reversed by treatment with HPYD through nasal administration. Our results indicate that HPYD may be a potential therapeutic candidate for the treatment of AD.

To further demonstrate that HPYD is a potential therapeutic for AD, we analyzed the mRNA levels of APP/PS1 transgenic mice to investigate the effect of HPYD on the brain. APP/PS1 transgenic model mice have early memory dysfunctions even before the degeneration of synapses and neurons (Dickey et al., 2003). The mRNA levels of many genes in APP/PS1 transgenic model mice are dysregulated, but are restored to normal levels after treatment with HPYD. The 119 genes that were differentially expressed were involved in inflammatory reaction, gluco-lipid metabolism and other pathways. Neuro-inflammation has been recognized as playing an important role in the pathogenesis of AD (Cacquevel et al., 2004; Sagy-Bross et al., 2013), which is mediated by microglia (MG) that can participate in the immune response, leading to increase in pro-inflammatory cytokines and chemokines (Lucin and Wyss-Coray, 2009; Prinz et al., 2011). Inflammasomes are responsible for the maturation and release of pro-inflammatory cytokines and the activation of an inflammatory form of cell death. NLRP3 inflammasome, one of the most widely studied members of the NLR family, can be activated by Aβ and enhances AD progression by mediating a detrimental chronic inflammatory tissue response (Heneka et al., 2013). Our results demonstrated that inflammasomerelated CASP1 and CASP4, which mediates non-canonical activation of the NLRP3 inflammasome, was highly expressed in APP/PS1 transgenic model mice, which was in concordance with previous studies (Schmid-Burgk et al., 2015). Inflammatory factors, like APOBEC3, CLEC4D, DDX3X, FCGR4, IFIH1, IRF7, NAIP2, NAIP5, CLEC5A, PTPRC and PLA2G4A, were also overexpressed in APP/PS1 transgenic model mice. PLA2G4A has been demonstrated to mediate apoptotic neuronal death in AD brain and could be induced by aggregated Aβ peptide1−42 (Sagy-Bross et al., 2013). PTPRC (CD45) was upregulated in microglial and has been association with AD (Masliah et al.,

#### REFERENCES

1991). In addition, we also observed that cell-cycle protein CDK1 (CDC2) was upregulated in APP/PS1 transgenic model mice, which is associated with the pathogenesis of AD (Johansson et al., 2005). Most importantly, our study also found that the disordered changes in neurons were effectively restored by treatment with HPYD through nasal administration, indicating HPYD as a potential therapeutic for AD.

In summary, we designed a new β-sheet breaker peptide HPYD, which showed better in vitro stability and inhibitory effects on Aβ1−42 aggregation compared to H102. HPYD could improve the learning and memory ability in APP/PS1 transgenic mice by reducing Aβ and APP protein levels. Microarray analyses demonstrated that dysregulated gene expression in model mice could be restored to normal levels after treatment with HPYD by inhibiting the aggregation of Aβ. This provides a novel therapeutic strategy for the treatment of AD.

#### AUTHOR CONTRIBUTIONS

SX conceived project, WL, FS, and SX designed the study and wrote the paper. WL and FS performed the experiments. MW, FJ, XB, and LL provided technical assistance and contributed to the preparation of the figures and manuscript. HT contributed to English editing and academic writing in the whole manuscript. All authors reviewed the results and approved the final version of the manuscript.

#### FUNDING

This work was supported by grants from Tianjin science and technology plan projects (no: 16YFZCSY01000), National Natural Science Foundation of China (no: 81602512) and China Postdoctoral Science Foundation (no: 2015M581307).

#### ACKNOWLEDGMENTS

We are grateful to Miss Qing Li from Tianjin cancer institute & hospital for her relevant critical revision of this manuscript.

in mild Alzheimer's disease patients. Alzheimers. Dement. 12, 110–120. doi: 10.1016/j.jalz.2015.06.1893

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Liu, Sun, Wan, Jiang, Bo, Lin, Tang and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Frontal Control Process in Intentional Forgetting: Electrophysiological Evidence

Heming Gao\*, Mingming Qi and Qi Zhang

School of Psychology, Liaoning Normal University, Dalian, China

In this study, we aimed to seek for the neural evidence of the inhibition control process in directed forgetting (DF). We adopted a modified item-method DF paradigm, in which four kinds of cues were involved. In some trials, the words were followed by only a forgetting (F) cue. In the other trials, after a word was presented, a maintenance (M) cue was presented, followed by an explicit remembering (M-R) cue or an forgetting (M-F) cue. Data from 19 healthy adult participants showed that, (1) compared with the remembering cue (i.e., M-R cue), forgetting cues (i.e., M-F cue and F cue) evoked enhanced frontal N2 and reduced parietal P3 and late positive complex (LPC) components, indicating that the forgetting cues might trigger a more intensive cognitive control process and that fewer amounts of cognitive resources were recruited for the further rehearsal process. (2) Both the M cue and the F cue evoked enhanced N2 and decreased P3 and LPC components than the M-R or M-F cue. These results might indicate that compared with the M-R and M-F cues, both the M and F cues evoked a more intensive cognitive control process and decreased attentional resource allocation process. (3) The F cue evoked a decreased P2 component and an enhanced N2 component relative to the other cues (i.e., M-R, M-F, M), indicating that the F cue received fewer amounts of attentional resources and evoked a more intensive cognitive control process. Taken together, forgetting cues were associated with enhanced N2 activity relative to the maintenance rehearsal process or the remembering process, suggesting an enhanced cognitive control process under DF. This cognitive control process might reflect the role of inhibition in DF as attempting to suppress the ongoing encoding.

Keywords: directed forgetting, maintenance rehearsal, cognitive control, P2, N2

# INTRODUCTION

Intentionally ignoring or forgetting out-of-date information is essential for memory function (Anderson et al., 2004; Anderson and Hanslmayr, 2014). The processing of task-relevant information may be disrupted by irrelevant information. Intentional forgetting might be helpful in reducing this interference (Nowicka et al., 2011; Benoit and Anderson, 2012). Intentional forgetting is usually investigated by adopting an item-method directed forgetting (DF) paradigm. During the study phase, remembering or forgetting cues are provided randomly following each item. To-be-remembered (TBR) items are followed by remembering cues, and to-be-forgotten (TBF) items are followed by forgetting cues. Generally, TBR items show superior memory performance over TBF items (Bjork and Woodward, 1973; Basden et al., 1993). This effect is called the DF effect.

Edited by:

Antonella Gasbarri, University of L'Aquila, Italy

#### Reviewed by:

Alexandra Key, Vanderbilt University Medical Center, United States Alfredo Meneses, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Mexico

> \*Correspondence: Heming Gao<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience

Received: 10 September 2017 Accepted: 29 December 2017 Published: 11 January 2018

#### Citation:

Gao H, Qi M and Zhang Q (2018) Frontal Control Process in Intentional Forgetting: Electrophysiological Evidence. Front. Neurosci. 11:757. doi: 10.3389/fnins.2017.00757

According to the selective rehearsal account, the DF effect is due to the selective rehearsal of TBR words (Basden et al., 1993; Sheard and MacLeod, 2005). If a remembering instruction is received, participants engage in an elaborate rehearsal. Successful intentional forgetting occurs owing to the passive decay of an unrehearsed memory trace (Bjork and Woodward, 1973; Basden et al., 1993; MacLeod, 1999). The attentional inhibition account argues that an active inhibition process is triggered by forgetting cues (Geiselman and Bagheri, 1985; Zacks et al., 1996). This inhibitory process might serve to cease the rehearsal process of TBF items or suppress the memory representation (van Hooff and Ford, 2011).

With the merit of high temporal resolution, the event-related potential (ERP) technique has been employed to explore the neural activity underlying DF (Paz-Caballero et al., 2004; van Hooff and Ford, 2011; Gao et al., 2016a). The P2 component has been associated with attentional allocation process, with enhanced attention resulting in increased P2 amplitudes (Thorpe et al., 1996; Bergström et al., 2007; Qi et al., 2016). Some DF studies found that a more positive frontal P2 component was evoked for remembering vs. forgetting cues (Cheng et al., 2012; Gao et al., 2016a), indicating that forgetting cues received fewer amounts of attentional resources. Some studies found that compared with remembering cues, forgetting cues evoked more positive ERPs over the frontal scalp but evoked less positive ERPs over the parietal scalp (Paz-Caballero et al., 2004; van Hooff and Ford, 2011). Recently, some studies found that forgetting cues evoked a more negative N2 over the frontal scalp but decreased P3 and late positive complex (LPC)components over the parietal scalp compared with remembering cues (Yang et al., 2012; Patrick et al., 2015; Gao et al., 2016a). These studies suggested that the enhanced frontal ERP activity associated with forgetting cues might reflect the attentional inhibition process.

In the typical item-method DF paradigm, participants are aware that each item has an equal possibility of being followed by a remembering or a forgetting cue. They do not engage in an elaborate rehearsal when the items are presented. These items are kept in working memory by rote rehearsal or maintenance rehearsal before cues are presented (Woodward et al., 1973; Greene, 1987). Therefore, different cognitive strategies might be adopted in response to different cues. Specifically, remembering cues trigger the elaborate rehearsal process for TBR items, while this process was absent for forgetting trials, in which TBF items are passively decayed or inhibited (Wylie et al., 2008). The ERP cue effect (remembering vs. forgetting cue) might reflect that participants adopted different cognitive strategies. There is no firm evidence showing that forgetting cues trigger the inhibition control process.

Previous studies have demonstrated that cognitive control over overt behavior is always associated with the activity of the frontal/prefrontal cortex. For example, this frontal activity was always observed in motor response inhibition tasks (e.g., Bokura et al., 2001; Aron et al., 2014) and switching tasks (e.g., Dove et al., 2000; Philipp et al., 2013). Goal-directed cognition generally requires cognitive control, and Anderson et al. (2004) suggested that an explicit forgetting instruction might place demands on controlled attention. Conway and Fthenaki (2003) found that the DF effect was diminished in patients with frontal lobe damage. Therefore, it seems that DF may involve the attentional/cognitive control process. Wylie et al. (2008) found that the neural activity associated with intentional forgetting differed from that associated with unintentional forgetting and intentional remembering, and they speculated that the maintenance rehearsal process is associated with the activity of the inferior frontal regions, and that the activity in the parahippocampal area may reflect the attempt to relate the maintained items to the remembering/forgetting instructions. However, no studies have investigated the neural activity associated with the maintenance rehearsal process in DF.

The present study focused on the neural activity of the maintenance rehearsal process. In the item-method DF procedure, the maintenance rehearsal process was interrupted by remembering and forgetting cues. According to the selective rehearsal account, maintenance rehearsal was passively ceased without any further cognitive processes acting upon the TBF information. However, the attentional inhibition account suggested that processing resources were actively withdrawn from the memory representation of TBF items, and attention was inhibited from returning to the memory representations of TBF items. Therefore, if DF is a passive process, decreased neural activity would be found for the forgetting process vs. the maintenance rehearsal process. On the contrary, if enhanced neural activity was found for the forgetting process vs. the maintenance rehearsal process, it might imply that the forgetting cues triggered an active inhibitory process.

In this study, we adopted a modified item-method DF paradigm, in which a maintenance (M) cue was presented before the remembering/forgetting cues (**Figures 1A,B**). Specifically, when participants saw the M cue, they could not know whether the word was TBR or TBF until the following cues (remembering M-R or forgetting M-F) appeared. Therefore, when the M cue was presented, the participants refreshed the words through maintenance rehearsal until the remembering/forgetting cue was presented. The M cues would trigger the maintenance rehearsal process. In the other trials, the word was followed by only a forgetting cue (F), and this word was categorized as a TBF item (**Figure 1C**). The F cues would trigger the DF process. By using this modified paradigm, we investigated the neural activity underlying maintenance rehearsal and DF (i.e., ERPs evoked by M and F cues).

Previous DF studies found that forgetting cues evoked more negative frontal N2 and less positive parietal P3 components compared with remembering cues (Patrick et al., 2015; Gao et al., 2016a,b). A similar ERP effect would be expected for the M-R vs. M-F cues in this study. As a subcomponent of the P3 component, P3a exhibits a fronto-central scalp distribution and has been associated with the reallocation of attentional resources. Therefore, an enhanced P3a would be found for the M-R cue relative to the M-F cue. In addition, it is necessary to note that the M cues always preceded the M-F/M-R cues, and encoding of item information might benefit from maintenance rehearsal, leading to greater memory trace strength for the M-R condition relative

to the M-F condition. Therefore, we speculated that the ERP difference between the M and M-F cues might be modulated by the differential memory trace strength of the words. To eliminate this order effect, the third condition (F) was designed.

time-locked to the cue onset.

This study mainly focused on the ERP differences between the M and F cues. Previous DF studies found that forgetting cue evoked a decreased frontal P2 component compared with remembering cue (Cheng et al., 2012; Gao et al., 2016a). Similarly, in this study, we predicted that an enhanced P2 component would be observed for the M cues relative to the F cues. We hypothesized that if the memory representation of TBF words was passively decayed, the maintenance rehearsal process would be decreased after the F cues were presented. Therefore, the maintenance rehearsal process triggered by M cues would be more intensive than that triggered by F cues. The parietal P3 and LPC components are associated with the memory rehearsal process (Patrick et al., 2015; Gao et al., 2016b). Accordingly, enhanced P3 and LPC activities would be found for the M cues relative to the F cues. However, if the F cues triggered an active inhibition process to the memory representation of TBF words, the forgetting process might be more effortful than the maintenance rehearsal process. The frontal N2 component is known to be related to executive control (Espinet et al., 2012), inhibitory control of task-irrelevant information (Getzmann et al., 2015; Iannaccone et al., 2015), and the inhibitory process for TBF items (Bergström et al., 2007; Mecklinger et al., 2009; Patrick et al., 2015; Gao et al., 2016a,b). Levy and Anderson (2008) suggested that mechanisms engaged in attentional inhibition might be relate to cognitive control processes that are similar to those used to control overt behavior. Therefore, an enhanced frontal N2 component would be evoked for the F cues relative to the M cues in this study.

# MATERIALS AND METHODS

#### Participants

Twenty undergraduate native Chinese college students took part in this experiment. Because of excessive artifacts in the electroencephalographic recording, one participant was excluded from the analysis (<50% trials were valid after artifact rejection). Therefore, data from 19 participants were included in the analyses (nine male and 10 female participants, mean age = 23.1 years, standard deviation = 1.85). All participants were righthanded and self-reported as healthy. All participants had normal or corrected-to-normal eyesight, and none were color blind. This study was approved by the Research Ethics Committee of Liaoning Normal University of China and was in accordance with the ethical guidelines of the Declaration of Helsinki. All participants have granted their written informed consent, and were paid on completion of the experiment.

#### Design and Materials

In the study phase, after the words were presented, the participants either received a maintenance cue followed by a remembering/forgetting cue (**Figures 1A,B**) or received only a forgetting cue (**Figure 1C**). Specifically, if a green cue (string of green Xs, cue 1) followed the word, the participants needed to see the following cue (cue 2) to judge this word as TBR or not. If a green cue followed (cue 2), it was a TBR word (**Figure 1A**); if a red cue followed (cue 2), it was a TBF word (**Figure 1B**). If a word was only followed by a red cue (cue 1), this word was a TBF word (**Figure 1C**), and no additional cues followed. Therefore, there were three conditions (three kinds of words): maintain-remember (M-R), maintain-forget (M-F), and forget (F). A within-subject design was used in this study. The three conditions were presented in a pseudo-randomized order, with

the constraint that no more than three consecutive trials could be from the same condition. The assignment of color to the remembering cue or forgetting cues was counterbalanced across subjects.

The learning materials were Chinese double-character nouns, which were selected from the top 8000 words in "The Modern Chinese Frequency Dictionary" with a mean frequency of 7.144 per thousand. Words were assigned to six lists, each containing 60 words. The mean number of strokes and frequency of the words were matched across different lists. Half the lists were used as learning materials for the study phase, and the remaining lists served as new words (distractors) for the test phase. Additionally, two buffer words followed by remembering cues were presented at the beginning and end of the study phase, which were excluded from subsequent analyses. Except for the buffer words, 180 words (60 words per condition) were presented in the study phase. The test phase consisted of 180 old words (60 of each from the M-R, M-F, and F words) and 180 new words (distractors). The sequence of presentation for list sets was counterbalanced across participants. The words were printed in black (RGB: 0, 0, 0), whereas the cues (Xs) were printed in green (RGB: 0, 255, 0) or red (RGB: 255, 0, 0). All stimuli (words and cues, font size 28 pt) were presented on a silver-gray background (RGB: 192, 192, 192). The participants sat approximately 80 cm from a computer screen.

In the test phase, a recognition test was conducted for the participants. Specifically, if a word had presented in the study phase (i.e., both TBR and TBF words), the participants were asked to give an "old" response, or else, give a "new" response.

#### Procedure

During the study phase, each trial began with a 250 ms fixation cross, followed by a 500 ms blank screen. Then, a word was presented for 500 ms. For the M-R or M-F condition, after a random blank screen of 1,300–1,700 ms, a maintenance cue (cue 1) appeared for 250 ms, followed by a 1,300–1,700 ms blank screen, then a remembering/forgetting cue (cue 2: M-R/M-F cue) appeared for 250 ms, followed by a 2,500 ms blank screen (**Figures 1A,B**); For the F condition, after a word was presented, a 1,300–1,700 ms blank screen was presented, then a forgetting cue appeared for 250 ms, followed by a 2,500 ms blank screen (**Figure 1C**).

In the test phase, each trial began with a 250 ms fixation cross, followed by a 500–800 ms blank screen, and then a word appeared for 1,500 ms. Next, a 1,000 ms blank screen was presented. The participants were asked to press "f " or "j" on the keyboard to make old/new responses to the word as quickly and accurately as possible. The key assignment for the old/new responses was counterbalanced among participants.

# Data Analysis

#### Behavioral Data

The old response rate was defined as the percentage of old responses in each condition (i.e., hit rates to M-R, M-F, and F words and false-alarm rates to foils). A repeated-measure analysis of variance (ANOVA) with the factor word type (M-R, M-F, F, new) was performed on the old response rate.

#### ERP Recording and Analysis

Brain electrophysiological activity was recorded from a 64- Channel EEG recording system (Brain Products, GmbH, Germany) with references on a central midline electrode. A vertical electrooculogram (EOG) was recorded with electrodes placed below the right eye. A horizontal EOG was recorded with electrodes placed on the right canthi. All interelectrode impedance was maintained below 5 k. EEG and EOG were amplified using a 0.05–100 Hz bandpass filter and continuously sampled at 500 Hz for off-line analysis.

Raw EEG data were processed offline using BrainVision Analyzer version 2.0 (Brain Products, GmbH; Gilching, Germany). For the data analysis, ERPs time-locked to the cues onset (during the study phase) were re-referenced algebraically to the average of the left and right mastoids. After ocular correction (Gratton et al., 1983), EEGs were digitally filtered with a 30 Hz low-pass filter with a 24 bit analog-to-digital converter. ERPs for all cues during the study phase were then segmented into 1,000 ms epochs surrounding stimulus presentation and baseline-corrected with respect to 200 ms pre-stimulus. Trials contaminated with EOG artifacts (mean EOG voltage exceeding ±80 µV) or those with artifacts due to amplifier clipping, bursts of electromyographic (EMG) activity, or peak-to-peak deflection exceeding ±100 µV were excluded from averaging. EEGs recorded in all conditions were averaged separately for each participant. The mean numbers of trials retained after artifact rejection were as follows, M-R cue: mean = 42.2, SD = 6.7, range, 32–52; M-F cue, mean = 42.8, SD = 5.6, range, 31–52; M cue: mean = 95.2, SD = 12.8, range, 78–119; F cue: mean = 47.4, SD = 7.4, range, 35–58.

For the study phase, the P2 (120–180 ms), N2/P3 (200– 400 ms), and LPC (500–800 ms) time windows were chosen for statistical analysis, which corresponded to the typical latency range of the P2 (Smid et al., 1999), N2 (Folstein and Van Petten, 2008), P3 (Polich, 2007), and LPC (Patrick et al., 2015; Gao et al., 2016b) components.

The grand-averaged ERPs (**Figure 3**) showed that the maximum ERP difference during the P2 epoch distributed over the fronto-central scalp. Preliminary inspection of the data indicated that there were no ERP differences between conditions in the P2 time window at parietal electrodes (P3, Pz, P4). Therefore, data from the anterior-central recording sites [three frontal electrodes (F3, Fz, F4) and three central electrodes (C3, Cz, C4)] were selected for statistical analysis and divided into three levels (left vs. middle vs. right) of the hemisphere. Repeatedmeasures ANOVAs with cue type (M, M-R, M-F, F), caudality (frontal, central), and hemisphere (left, middle, right) as withinsubject factors were performed on the mean amplitudes during the 120–180 ms time period.

As shown in **Figure 3**, during 200–400 ms, the M and F cues evoked a frontal N2 component, whereas the M-R and M-F cues evoked a frontal N2/P3 complex. The N2/P3a was maximally recorded at frontal sites around 300 ms (Campanella et al., 2002). Therefore, the selected epoch corresponded to the typical latency range and scalp distribution of the N2/P3 complex. Additionally, M-R/M-F cues evoked a parietal P3 component during 200–400 ms. Therefore, data from the anterior-posterior recording sites [three frontal electrodes (F3, Fz, F4), three central electrodes (C3, Cz, C4), and three parietal electrodes (P3, Pz, P4)] were selected for statistical analysis and were factorized into three levels (left, middle, right) of the hemisphere. Repeated-measures ANOVAs with cue type (M, M-R, M-F, F), caudality (frontal, central, parietal), and hemisphere (left, middle, right) as withinsubject factors were performed on the mean amplitudes during the 200–400 ms period.

During the LPC (500–800 ms) time window, data from the anterior-posterior recording sites (F3, Fz, F4, C3, Cz, C4, P3, Pz, P4) were factorized into three levels (left, middle, right) of the hemisphere. Repeated-measures ANOVAs with cue type (M, M-R, M-F, F), caudality (frontal, central, parietal), and hemisphere (left, middle, right) as within-subject factors were performed on the mean amplitudes during the 500–800 ms period.

For the ERP data, to avoid describing large amounts of statistical data concerning scalp distribution effects, only main effects or interactions that included the cue factors were reported. All effects with >1 ◦ of freedom were adjusted for sphericity violations by using the Greenhouse-Geisser correction. Main effects (or interactions) were subjected to Bonferroni-corrected pairwise comparisons (or simple effect test).

### RESULTS

#### Behavioral Results

The old response rate was 75.5% for the M-R words, 59.3% for the M-F words, 52.1% for the F words, and 17.0% for the new words. The ANOVA results revealed a main effect of word type on the old response rate, F(3, 54) = 134.40, p < 0.001, η 2 p = 0.882. Pairwise comparisons revealed that the old response rate was higher for M-R words than for all other words, ps < 0.001, ds ≥ 1.41. The old response rate was higher for M-F words relative to F words and new words, ps ≤ 0.001, ds ≥ 0.50; the old response rate was higher for F words than for the new words, p < 0.001, d = 2.66 (**Figure 2**).

#### Electrophysiological Results

During the P2 (120–180 ms) time window, the ANOVA results revealed a main effect of cue type, F(3, 54) = 5.295, p = 0.003, η 2 p= 0.226. Pairwise comparisons revealed that the F cue evoked smaller P2 amplitudes than the other types of cues (M-R, M-F, M), ps ≤ 0.027, ds ≥ 0.32. No interactions that included the cue factor were found, ps > 0.378.

During the 200–400 ms time window, the results revealed a main effect of cue type, F(3, 54) = 51.90, p < 0.001, η 2 p = 0.742.  Simple effect analyses revealed that the M-R cue evoked more positive ERPs relative to the other cues (M-F, M, F) over all scalps, ps ≤ 0.001, ds ≥ 0.39; the M-F cues evoked more positive ERPs relative to the M and F cues over the whole scalps, ps ≤ 0.004, ds ≥ 0.62; and the M cues evoked more positive ERPs relative to the F cues over all scalps, ps ≤ 0.019, ds ≥ 0.26.

During the LPC epoch (500–800 ms), the Cue type × Caudality × Hemisphere interaction was significant, F(12, 216) = 1.91, p = 0.035, η 2 p = 0.096. Simple effect analyses revealed that the M-R cues evoked more positive ERPs relative to M cues at central-parietal scalp electrodes (C3, Cz, C4, P3, Pz, P4), ps ≤ 0.028, ds ≥ 0.88; the M-F cues evoked more positive ERPs relative to M cues at central-parietal scalp electrodes (C3, Cz, C4, P3, Pz, P4), ps ≤ 0.048, ds ≥ 0.89; the M-F cues evoked more positive ERPs relative to F cues at C3 electrodes, p = 0.014, d = 0.98; and the M cues evoked less positive ERPs relative to the F cues at Pz and P4 electrodes, ps ≤ 0.026, ds ≥ 0.78.

#### Correlational Analyses

To better determine the functional means of observed ERP effects, Pearson correlation analysis was performed to investigate the association between the recognition accuracy (old response rate for M-R, M-F, and F words) and the amplitudes of the frontal N2 or the parietal P3 and LPC components. The amplitudes of the frontal N2 component were calculated by averaging the amplitudes from three frontal electrodes (i.e., F3, Fz, F4), as well as from three parietal electrodes (i.e., P3, Pz, P4) for the P3 and the LPC amplitudes. The results showed a positive correlation between the accuracy and the fronto-central N2 amplitudes, r = 0.544, p < 0.001. A positive correlation was found between the accuracy and the parietal P3 amplitudes, r = 0.409, p = 0.002. No correlation was found between the accuracy and the parietal LPC amplitudes, r = 0.09, p = 0.505.

## DISCUSSION

The aim of the present study was to investigate whether forgetting is an active process that is more effortful than the maintenance rehearsal process. To trigger the maintenance rehearsal process, a maintenance cue was added into the item-method DF paradigm. The main findings are as follows: Consistent with previous DF studies, a typical DF effect was found. Memory performance benefited from the maintenance rehearsal process. ERP timelocked to the cues indicated that two continuous stages were involved in the forgetting process: the attentional withdrawal process, which was reflected by decreased frontal P2 activity, and the attentional inhibition process, which was reflected by increased frontal N2 activity. The enhanced frontal ERP activity associated with the forgetting process relative to the maintenance rehearsal process suggested that forgetting is an active process.

#### Behavioral Results

The memory performance was superior for the studied words (M-R, M-F, F) relative to the false-alarm rate for foils, indicating that all the studied words were reasonably encoded during the study phase. Consistent with previous DF studies (Bjork and Woodward, 1973; Basden et al., 1993), a typical DF effect was observed, with higher recognition performance for M-R words relative to M-F words, indicating an enhanced encoding process for M-R words during the study phase. This DF effect demonstrated that participants had successfully manipulated the words according to different (remembering, forgetting) instructions.