Humanin Specifically Interacts with Amyloid-β Oligomers and Counteracts Their in vivo Toxicity

INTRODUCTION

Alzheimer’s disease (AD) is the most common form of progressive senile dementia, affecting a large proportion of aged people in developed countries and causing memory impairment, disordered cognitive function and decline in language abilities [1]. Deposition in several brain areas of amyloid plaques mainly composed of amyloid-β (Aβ) peptides, and intra-cellular neurofibrillary tangles of hyperphosphorylated tau are the central neuropathological features of AD [2]. Studies aimed at deciphering the relationship between plaque burden and neuronal death indicate that oligomeric pre-fibrillar assemblies of Aβ, more than fibrils, play a key role in the toxicity and early cognitive impairment in AD [2–5]. Despite efforts to develop disease-modifying molecules, no effective therapy is yet available for AD. Numerous strategies have been designed aimed at preventing or delaying the self-assembly of monomeric Aβ into oligomeric forms, including molecules that interact directly with the misfolded protein, modify the kinetics of fibril formation, and facilitate their re-absorption [6–8].

Peptide-based inhibitors that interact directly with Aβ and interfere with the formation and/or biological properties of Aβ aggregates have been proposed as therapeutic candidates. The 24-residue peptide humanin (HN), first described as a neuroprotective factor identified from the cDNA from a protected lobe of an AD patient’s brain [9], has stimulated interest on account of its potential as an endogenous protective molecule.

HN is a mitochondrial-derived peptide, physiologically expressed in various organs and highly conserved across species, from the nematode Caenorhabditis elegans to humans, which may act as a retrograde signaling molecule to transmit information from the mitochondria to the nucleus and beyond [10]. This peptide is cytoprotective against a variety of stress and disease conditions, including AD, and low circulating HN levels may represent a risk for the insurgence of age-related pathologies [10, 11].

At central level, the neuroprotective action of HN was mediated by different mechanisms, involving the ability to prevent the aggregation and deposition [12, 13] of Aβ and to counteract its neurotoxic effects. HN can also bind to its putative receptor activating the intracellular signaling cascade and inhibiting c-Jun N-terminal kinase [14]. Studies in vitro have shown that HN acts as an anti-oxidant molecule, counteracting the Aβ-induced oxidative stress [15, 16]. It also protects neuronal cells from apoptosis induced by the amyloid-β protein precursor (AβPP) by binding to the apoptosis-regulator protein Bax [14].

Structure-function relationship studies showed that the amino acid residues between the Pro3 and Pro19 of HN were particularly important for its neuro- and cyto-protective activities [9]. On this basis a new synthetic peptide was developed in which Gly was substituted for Ser14 (HNG), proving about 1000 times more effective than HN on neuronal cell death in vitro [14]. When chronically administered to transgenic mice overexpressing both AβPP and mutant human presenilin-1, HNG significantly reduced the neuronal Aβ deposits [17–19]. All these findings, upholding the neuroprotective effect of endogenous HN and HNG, support their use as peptide-based inhibitors.

To clarify the underlying mechanisms better, we applied a multidisciplinary and integrated approach to investigate the ability of HN and HNG to interact directly with Aβ₄₂ and interfere with the formation and toxicity of oligomers. Experiments in cell-free conditions indicated that HN and HNG have similar effects on Aβ aggregation, interacting with preformed toxic soluble oligomeric species. In vivo studies were also done to investigate how HN and HNG counteracted the toxicity specifically due to Aβ oligomeric species. We used transgenic CL4176 C. elegans, in which the inducible expression of human Aβ₄₂ results in the specific accumulation of oligomeric assemblies in the body wall muscle cells which leads to paralysis [20–23]. This strain has been widely employed as a simplified invertebrate prototype of Aβ amyloidogenesis, and is a valuable in vivo animal model to elucidate the pharmacological mechanisms involved in the protective action of some anti-Aβ compounds [20, 25]. HN and HNG similarly reduced soluble oligomers, and protected nematodes against the Aβ toxicity. However, while HN specifically decreased the oligomers by promoting their assembly into larger aggregates, the main in vivo effect of HNG was the reduction of total Aβ levels. These data indicate for the first time the relationship between the amino acid sequence of HN-derived peptides and their mechanisms of action.

MATERIALS AND METHODS

RESULTS

Circular dichroism studies were done first to investigate the secondary structure of HN and HNG. Peptides were dissolved in water at the final concentration of 40 μM, put into a 0.1 mm path-length quartz cuvette and the spectra were obtained at 25°C by scanning the far-UV region between 190–260 nm at 20 nm/min with a response time of 0.5 s. After averaging six accumulations, the spectrum of the buffer was subtracted, the mean residue molar ellipticity calculated and the spectrum smoothed using a regularization method [40]. The spectra of both peptides were superimposable with strong negative dichroism around 200 nm (Fig. 1), indicating that the structure is mainly random, which is in line with published data [41, 42] and suggests the absence of any β-sheet containing aggregates, which occur at higher peptide concentrations [43]. The absence of β-sheet was also confirmed by the lack of any ThT signal upon incubation of 2 μM HN or HNG (data notshown).

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Fig.1

Circular dichroism (CD) spectra of HN and HNG. Polypeptides were dissolved at the final concentration of 40 μM in water. CD spectra are the average of six scans. Data are mean residue molar ellipticities (MRE). The two spectra gave a strong negative band around 200  nm, indicative of a low degree of ordering in the monomer.

This allowed us to investigate the effects of HN and HNG on the Aβ aggregation kinetics in cell-free system using a ThT assay. A 24-amino acid fragment of the rat albumin (rAlb_25-48) has been employed as negative control. Equimolar concentrations of HN or HNG, added to 2 μM Aβ₄₂ solution, similarly delayed fibril formation and halved the maximal ThT signal (Fig. 2A and Table 1) suggesting their ability to reduce the amount of Aβ fibrils and/or change their structure. This effect was specific for HN and HNG since 2 μM rAlb_25-48 did not modify the aggregation kinetics of Aβ₄₂ peptide (Fig. 2 and Table 1). To further quantify these effects, the ThT traces were normalized (Fig. 2B) and the half time of transition t_1/2 (i.e., when half the maximum ThT signal is reached), and the growth rate of fibril formation were calculated. HN and its derivative similarly and significantly increased t_1/2 and reduced the fibril growth rate (Table 1).

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Fig.2

Effects of HN and its derivative on the kinetics of Aβ₄₂ fibril formation. A) Two-μM Aβ₄₂ were dissolved in 50  mM sodium phosphate, pH 7.4, with or without 2 μM HN, HNG or rAlb_25-48 and 20 μM thioflavin T (ThT). The time course of changes in ThT fluorescence intensity are shown, as arbitrary units (A.U.). The results of three independent experiments are shown. B) Data in A) after normalization on the maximal ThT signal. Data are the mean±SD, n = 3. **p < 0.0001 versus Aβ₄₂ alone according to one-way ANOVA and Bonferroni’s post hoc test.

We investigated the ability of HN and HNG to interfere with Aβ₄₂ oligomerization with an SPR-based immunoassay, developed to specifically recognize transient and biologically relevant oligomers [30]. Oligomers bind to chip-immobilized 4G8 antibody in a pseudo-irreversible manner (very slow dissociation, probably due to multivalent binding), while monomers bind but rapidly dissociate, and higher-order aggregates show no SPR signal [30].

This assay is useful to test potential anti-oligomeric compounds [30]. In initial studies, we added the peptide to a freshly prepared Aβ₄₂ solution and recorded the oligomer-dependent SPR signal after 5 h incubation (Fig. 3A) [30]. Both HN and HNG inhibited this signal (Fig. 3B) and the effect was dose-dependent (Fig. 3C). IC₅₀ were 6.9 μM (5.5–8.5 CI) for HN and 10.2 μM (8.8–12.0 CI) for HNG. This inhibitory effect could be ascribed only to the interaction of HN and its derivative HNG with Aβ₄₂, since the two peptides did not bind immobilized 4G8 (Supplementary Figure 3).

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Fig.3

Effects of HN and HNG on Aβ₄₂ oligomers detected by a surface plasmon resonance-based immunoassay. A) In the first experimental protocol synthetic monomeric Aβ₄₂ (100 μM) was incubated at 25°C for 5 h with or without different concentrations of HN or HNG (0.3–100 μM), diluted 100-fold then flowed onto 4G8 immobilized on the SPR sensor chip. B) Representative sensorgrams obtained by flowing Aβ₄₂ solutions with or without 100 μM HN or HNG, for 2  min (bars). The signal at the end of the dissociation phase (i.e. at 780 s)is indicative of the oligomer-dependent SPR signal. C) Dose-response effects of HN and HNG on the Aβ₄₂ oligomer-dependent SPR signal. Data are percentages of the maximum SPR signal in the absence of inhibitors. D) In the second experimental protocol synthetic monomeric Aβ₄₂ (100 μM) were pre-incubated at 25°C for 5 h to form oligomers, HN or HNG (0.3–100 μM) were then added, and after 30  min the solutions were diluted 100-fold and flowed onto 4G8 immobilized on the SPR sensor chip. E) Representative sensorgrams obtained by flowing Aβ₄₂ solution with or without 100 μM HN or HNG for 2  min (bars). F) Dose-response effects of HN and HNG on the Aβ₄₂ oligomer-dependent SPR signal. Data are percentages of the maximum SPR signal in the absence of inhibitors.

Similar SPR experiments were performed with the control peptide rAlb_25-48. To this end, freshly prepared Aβ₄₂ was incubated with the control peptide, and after 5 hours of incubation, the solution was injected over immobilized 4G8 (Supplementary Figure 4). Data showed that rAlb_25-48 did not have any significant effect on Aβ₄₂ oligomers formation, thus confirming the specificity of HN and HNG in their anti-oligomeric activities.

Since this inhibition could be due to an effect either on oligomer formation or on oligomer binding to immobilized 4G8, we ran additional studies in which HN and HNG were added to preformed oligomers (Fig. 3D). In this case too, HN and HNG reduced the oligomer-dependent SPR binding signal (Fig. 3E), in a dose-dependent manner, with IC₅₀ 14.5 μM (95% CI 11.1–18.9) for HN and 11.9 μM (9.3–15.2) for HNG (Fig. 3F). HN was twice as active when added at the beginning of the aggregation than when added to preformed oligomers (6.9 versus 14.5 μM, p < 0.01, extra sum-of-squares F-test) suggesting that it either i) interferes with oligomer formation and/or ii) both act on preformed oligomers (in this case preventing their binding to immobilized 4G8). In contrast HNG gave similar IC₅₀s, indicating interactions with preformed oligomers only. Hypothetically this effect may be due to the ability of HN and HNG to shield hydrophobic residues responsible for the interaction of oligomers with immobilized 4G8. Otherwise, HN and HNG might favor the oligomer’s aggregation into larger assemblies, which are not sensed by the SPR assay.

To test this latter possibility, AFM, TEM and turbidity analysis were done. Incubation of 100 μM Aβ₄₂ at 37°C for 2 h resulted in the appearance of short scattered protofibrils (95–155 nm long) (Fig. 4A, B). HN co-incubated with Aβ₄₂ induced assembly of the short protofibrils into dense agglomerates which could be seen with TEM (Fig. 4D) but not AFM (Fig. 4C). This might be due to the different electrostatic properties of the agglomerates and oligomers, which did not allow their immobilization on the negatively charged mica surface [30] (Fig. 4C). Results were similar when Aβ₄₂ was incubated with HNG (Fig. 4E, F).

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Fig.4

Atomic force microscopy (AFM) and transmission electron microscopy (TEM) analysis. Synthetic Aβ₄₂ (100 μM) was incubated at 37°C for 2 h with or without 100 μM HN or HNG. Samples were then diluted ten times with 10  mM phosphate buffer, pH 7.4. Representative (A, C, E) AFM and TEM (B, D, F) images of Aβ₄₂ alone (A-B) and Aβ₄₂ co-incubated with HN (C-D) or HNG (E-F). Scale bar: 2 μm×2 μm scan for AFM images and 500  nm for TEM images.

Aggregation of Aβ₄₂ can also be assessed by turbidity at 400 nm. As shown in Fig. 5 the absorbance of 100 μM Aβ₄₂ incubated for 2 h at 37°C increased in the presence of 100 μM HN and HNG. These findings confirmed the presence of larger aggregates as suggested by TEM analysis.

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Fig.5

Turbidity analysis. Synthetic Aβ₄₂ (100 μM) in 50  mM phosphate buffer (pH 7.4) was incubated at 37°C for 2 h with or without 100 μM HN or HNG. Turbidity was determined by measuring the absorbance of samples at 400  nm. Data are the mean±SD, n = 3. **p < 0.0001 versus Aβ₄₂ alone according to one-way ANOVA and Bonferroni’s post hoc test.

We further investigated the ability of HN and its derivative to interfere with Aβ₄₂ oligomers in vivo using C. elegans as the animal model of Aβ-induced toxicity. We employed the transgenic CL4176 strain, in which the paralysis phenotype is specifically caused by the deposition of oligomeric Aβ₄₂ in the body muscle cells and no fibrillar amyloid aggregates are formed [20, 37]. After synchronization of the eggs, worms were placed at 16°C on NGM plates seeded with OP50 E. coli for 60 h and Aβ₄₂ expression was induced by raising the temperature to 24°C. The worms were given increasing concentrations of HN and HNG (5–250 μM) 12 h after the temperature rise and their paralysis was scored 24 h later. Both peptides protected CL4176 worms from paralysis in a dose-dependent manner, with similar IC₅₀ (31.9±1.5 μM for HN and 32.6±1.9 μM for HNG, mean±SE) (Fig. 6).

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Fig.6

Dose-response effects of HN and HNG on Aβ₄₂-induced paralysis in transgenic C. elegans. Egg-synchronized CL4176 worms were placed at 16°C on fresh NGM plates seeded with OP50 E. coli and the temperature was raised 60 h after plating. Twelve h later, worms were fed HN or HNG (5–250 μM) and the number of paralyzed worms was scored after 24 h. Data are percentages±SE of paralyzed worms compared to vehicle-treated ones (100 worms/group, three independent assays).

To clarify the mechanisms of the in vivo protective activity of HN and HNG we investigated their effects on Aβ expression/degradation and oligomerization. Dot blotting and western blotting experiments were carried out with lysates from transgenic CL4176 worms fed 50 μM HN or HNG for 24 h. Lysates from CL802 nematodes, which did not express Aβ, were used as negative controls [23]. At this dose HN reduced the worms’ paralysis by 27% (80.8±2.1 of paralyzed worms for control and 53.9±6.3% for HN-fed worms, p < 0.01, one-way ANOVA) and HNG by 30% (50.8±1.4, p < 0.01 versus control, one-way ANOVA). The same concentration of polypeptides had no effects in transgenic CL802 control worms (data not shown).

To investigate the effect of HN and HNG on Aβ oligomers, A11 and OC conformation-selective antibodies recognizing different classes of quaternary structure were used [39]. Results from dot-blot analysis indicated that HN and HNG similarly reduced the A11- immunoreactive signal by about 80% and OC-reactive oligomers by 20% (Fig. 7C-F). Worms treated with HNG, but not HN, also showed a 58% reduction of the total Aβ levels (Fig. 7A, B). In line with the dot blotting analysis, western blot analysis indicated an overall reduction of Aβ levels in the lysates of worms fed HNG accompanied by a significant reduction of the 20 kDa low-molecular weight and >48 kDa high-molecular weight oligomers bands, and 4 kDa band (Figs. 7 and 8).

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Fig.7

Effects of HN and HNG on total and oligomeric Aβ: dot-blot analysis. Representative dot blot of A) total Aβ (WO2), C) A-11 immunoreactive oligomers and E) OC immunoreactive assemblies in CL4176 transgenic worms fed vehicle or 50 μM HN or HNG for 24 h. CL802 transgenic worms were used as negative control. Equal amounts of protein from worm lysates (20 μg) were spotted in triplicate. Synthetic Aβ₄₂ (0.1 μg) was spotted as positive control. Total proteins on the blotted membranes were stained using 0.1% Ponceau Red solution and were used to normalize the immuno-specific signal for protein loading. The mean volume of the B) WO2 Aβ-immunoreactive, D) A11 oligomer-immunoreactive and F) OC immunoreactive spots were determined using Quantity One 1-D software. Immunoreactivity from three different independent samples, analyzed in triplicate, was expressed as the mean volume of the immunoreactive band/ total proteins±SE. **p < 0.001 versus CL4176 worms fed vehicle (one-way ANOVA).

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Fig.8

Effects of HN and HNG on total and oligomeric Aβ: western blot analysis. Representative western blot of Aβ species. CL4176 worms fed vehicle or 50 μM HN or HNG for 24 h. CL802 transgenic worms were used as negative control. Equal amounts of protein (50 μg) were loaded on each gel lane and immunoblotted with anti-Aβ antibody (6E10) or actin. Arrows indicate the Aβ monomers (4kDa), low-molecular weight oligomers (∼20 kDa) and high-molecular weight oligomers (from 48 up to 100 kDa). A) Image obtained 3 minutes after membrane exposure, which was the optimal time for visualization of the 20 kDa and >48 kDa bands, corresponding to Aβ low-molecular weight oligomers and high-molecular weight oligomers, respectively. B) Image obtained 8 minutes after membrane exposure which represented the optimal time for visualization of the Aβ monomers at 4 kDa. C) Immunoreactivity of 6E10 for the quantification of the monomeric 4 kDa band was done on the image obtained 8 minutes after membrane exposure. The low-molecular weight oligomers (∼20 kDa) and high-molecular weight oligomers (>48 kDa) bands were quantified on the membrane exposed for 3  min. Data are the mean volume of the immunoreactive band/ total proteins±SD obtained from three different independent samples, analyzed in triplicate. **p < 0.01 versus CL4176 worms fed vehicle (one-way ANOVA).

Western blot analysis indicated that HN significantly reduced the signal of the band at ∼20 kDa, corresponding to Aβ low-molecular weight oligomeric species, and concomitantly boosted the signal of the bands equal or higher of 48 kDa (Fig. 8). These effects were accompanied by a significant reduction of the level of 4 kDa monomeric Aβ (Fig. 8). Moreover, due to the specific ability of CL4176 C. elegans strain to produce only oligomeric Aβ₄₂ [20], the effect of HN was not accompanied by the formation of fibrillar amyloid aggregates. These findings suggest that HN counteracted Aβ-induced toxicity by thus promoting their assembly into larger, not fibrillar, assemblies.

The ability of HNG to lower Aβ levels in vivo has already been reported [18], and was ascribed to the ability of this compound to activate Aβ-degrading enzymes, particularly the neutral neuropeptidease neprilysin [18]. The structure and function of these enzymes are well conserved from mammals to C. elegans [44]. To investigate whether the reduction of total Aβ in CL4176 worms fed HNG was associated to neuropeptidase activation, the level of expression of neprilysin was evaluated. Results from dot-blot analysis indicated that HNG significantly increased the neprilysin immunoreactive signal from 6.12±0.3 neprylisin/μg protein to 24.02±2.9 neprylisin/μg protein whereas HN did not modify it (9.27±2.0 neprylisin/μg protein) (Fig. 9). These data suggest that the HNG-induced reduction of the total Aβ level in worms is likely the consequence of the HNG-induced overexpression of the Aβ-degrading enzyme.

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Fig.9

Effects of HN and HNG on neprilysin level. A) Representative dot-blot of neprilysin in CL4176 transgenic worms fed vehicle, 50 μM HN or HNG for 20 h. Equal amounts of protein from worm lysates (20 μg) were spotted in triplicate. Total proteins on the blotted membranes were stained using 0.1% Ponceau Red solution and were used to normalize the immuno-specific signal for protein loading. B) The mean volume of the neprilysin-immunoreactive spots were determined using Quantity One 1-D software. Immunoreactivity obtained from three different independent samples, analyzed in triplicate, was expressed as the mean volume of the immunoreactive band/μg proteins±SD. **p < 0.001 versus CL4176 worms fed vehicle (one-way ANOVA and Bonferroni’s post hoc analysis).

DISCUSSION

The protective effects of HN and HNG against Aβ-induced toxicity have been widely investigated in cells and animal models [13, 42]. Studies performed in cell free conditions showed that HN and HNG similarly interact with Aβ_1-40, affecting its folding, [12, 13]. Our present in vitro studies confirm and extend the above observations, demonstrating that HN and HNG directly interact with Aβ₄₂ oligomers, interfering with their toxic activities.

There is increasing evidence of the key role of Aβ soluble oligomers in the pathogenic process of AD, suggesting that small molecules, inhibiting Aβ self-oligomerization or interacting with it might be useful as future pharmacological treatments. We here examined for the first time the potential anti-oligomeric activity of HN and HNG. Quantitative results from SPR experiments indicated that HN and HNG bind to formed soluble Aβ oligomeric intermediates. Qualitative analysis using AFM and TEM, andquantitative data from turbidity assay showed that both polypeptides, similarly to tetracyclines, antibiotics with known anti-aggregating and anti-oligomeric activities [45], promoted the further aggregation of oligomers into larger assemblies with amorphous structures [45]. Similar amorphous structures have been already observed by Zou P et al. after incubation of Aβ_1-40 with HNG [13, 46]. This mechanism is presumably responsible for the effects of HN and HNG in hindering the formation of Aβ₄₂ fibrils and the generation of amyloid structuresin vitro. HN and HNG showed similar potencies, indicating that the substitution of Gly for the Ser14 in the HN sequence was probably not important for the interaction with Aβ oligomers.

These observations were confirmed in vivo in experiments in which HN and HNG counteracted the toxicity caused by Aβ₄₂ oligomers in CL4176 transgenic C. elegans. Transgenic worms expressing human proteins have been widely used to elucidate the molecular pathways underlying proteinmisfolding diseases [47, 48]. Moreover, they offer the unique opportunity to test the effect of potential therapeutic compounds on whole animals avoiding ethical problems and at the speed and cost of the in vitro cell-based assays. However, it is important to underlie that C. elegans is a simple multicellular organism avoid of a circulatory and central nervous system and that the findings obtained applying this model, although scientifically relevant, need to be validated in vertebrate animals and translation to human pathology requires caution.

HN and HNG showed similar protective effects against Aβ toxicity, with comparable IC_50, and we suggest that this is due to their ability to reduce the amount of toxic oligomers in vivo. Notably, whereas HN specifically reduced the formation of low-molecular weight oligomers favoring their assembly into larger assemblies, the overall decrease of low- and high-molecular weight oligomers caused by HNG is mainly due to the decrease of total Aβ levels. The ability of HNG to lower Aβ levels in vivo, without affecting APP production and secretase activities, has already been reported [18], and was ascribed to the HNG-induced activation of Aβ-degrading enzymes, particularly the neutral neuropeptidease neprilysin [18]. The structure and function of these enzymes are well conserved from mammals to C. elegans [44] allowing us the evaluation of neprilysin involvement in our model. We observed that, similarly to rodents, the reduction of total Aβ in CL4176 worms fed HNG was associated to the increase of neprilysin level.

The two polypeptides showed similar protective effects, with IC₅₀ comparable to those of other compounds which also had anti-oligomeric activity inC. elegans, such as tetracyclines. The pre-clinical and clinical anti-amyloidogenic activity of tetracyclines was studied and confirmed by independent research groups [20, 49–52].

In conclusion, both HN and HNG interact directly with Aβ₄₂ in vitro and interfere with the formation and toxicity of its oligomers. Moreover, they similarly protect transgenic C. elegans against the toxicity specifically induced by Aβ oligomers deposition. Here we show for the first time that HN counteracts the formation of oligomers in vivo by promoting their assembly into larger aggregates, whereas the reduction of oligomers caused by HNG can be ascribed to its ability to reduce the overall Aβ levels.

enlargethispage *2ptIn addition to AD, Aβ amyloid misfolding and deposition is a key feature for the development of inclusion body myositis (sIBM). In this progressive skeletal musculoskeletal pathology, frequently found in older subjects, Aβ deposits accumulated in skeletal muscle cells causing functional neuromuscular dysfunctions [53, 54]. Although sIBM involves an inflammatory process, it does not respond to anti-inflammatory drugs thus remaining an untreatable disease. An effective therapeutic strategy may be represented by the targeting of Aβ. Transgenic C. elegans strains overexpressing human Aβ in muscle cells, like CL4176 strain, have been widely used as good experimental models also for studies aimed at developing new therapeutic approaches for sIBM [53, 54]. In fact, although different tissues are affected in AD and IBM, the functional neuromuscular synaptic dysfunction caused by Aβ amyloidogenic assemblies is a common feature of the two diseases [21, 54]. The findings obtained indicate that HN and HNG may be relevant for the design of future beneficial pharmacological strategies for sIBM, too.