Development of GMP‐1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease

Abstract Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aβ) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria‐destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP‐1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP‐1 treatment of SH‐SY5Y cells results in decrease in mitochondria‐associated APP and protects SH‐SY5Y cells from toxic effect of Aβ1‐42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP‐1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.

peptide whereas more C-terminally located domain enriched with acidic amino acids acts as translocation arrest sequence. 11 Upon mitochondrial entry, APP forms stable translocation intermediate complexes with mitochondrial translocase of the outer membrane (TOM) as well as links together translocases the outer and the inner membranes. 4,11 Complexes between mitochondrial protein translocases and APP were particularly abundant in the affected areas of the brains of patients with AD suggesting connection between mitochondrial APP accumulation and AD pathology in the brain. 4 APP accumulated in the mitochondrial import channels can be degraded by several mitochondrial proteases including HtrA2/Omi and gamma secretase. 5,12 At present, factors contributing to mitochondrial APP accumulation under AD conditions are not known. Genetic variations in components of mitochondrial protein translocase can result in lower efficiency of mitochondrial protein import and APP accumulation in the import channels. 13 AD-associated mutations in the mitochondrial protein degradation system 14 can shift the APP uptake/ degradation balance towards mitochondrial APP accumulation. Different environmental factors can also contribute to the mitochondrial APP accumulation. For example, high intracellular cholesterol level, a risk factor for development of Alzheimer's disease, has been shown to affect APP secretion, post-translational modification and intracellular trafficking. 15 Although the molecular determinants for Ab mitochondrial import are not known, mitochondrial protein translocase also participates in the import of Ab peptide 16 suggesting that reduction in APP and Ab mis-targeting can promote neuronal survival under AD conditions. Nuclear-encoded proteins destined to mitochondria enter the organelle via the TOM complex consisting of protein-conducting channel formed by Tom40, two primary receptors, Tom20 and Tom70, the central receptor Tom22 and several adaptor subunits of small molecular mass. 17 The majority of mitochondrial precursor proteins enter the organelle via Tom20 receptor whereas hydrophobic mitochondria-destined proteins with internal targeting signals preferentially follow Tom70 receptor pathway. 17 Cytosolic molecular chaperones Hsp70 and Hsp90 as well as other specific factors in cytosol participate in targeting of newly synthesized proteins to the Tom70 receptor. 18,19 The interaction is mediated by the short C-terminal acidic peptide-EEVD identical in Hsp70 and Hsp90 which binds to the tetratricopeptide repeat motif (TPR) of Tom70 receptor by dicarboxylate clamp mechanism. 19

| Chemicals
All common chemicals were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. Innova Biosciences). C90 peptide was dissolved in HEPES-KOH buffer pH 7.2 at concentration 1 mg/mL and incubated with equimolar amounts of activated HRP for 5 minutes at room temperature. Reaction was quenched with 0.1 mol/L Tris-HCl, pH 7.5 for 10 minutes, and the products were passed through PD-10 columns (GE Healthcare, Uppsala, Sweden).

| Cloning and purification of human TPR
proteins Tom70, Tom34, FKBP51 and PP5 Full-length cDNA clones for respective proteins were obtained from IMAGE consortium (SourceBioScience, UK) and PCR amplified with forward and reverse primers containing overhangs with appropriate restriction sites and cloned into pGEX6 vectors (GE Healthcare, Uppsala, Sweden). Full-length Tom34, FKBP51 and PP5 proteins as well as cytoplasm-exposed domain of Tom70, amino acids 65-608, were amplified. Inserts were sequence verified, and the plasmids were  and protease inhibitor cocktail were added to prevent proteolysis.
The suspension was centrifuged for 30 minutes 50 000 9 g to remove cell debris, and supernatant was loaded onto 1 mL Gst-trap 4B column (GE Healthcare Uppsala, Sweden). After column washing with 30 mL PBS Gst fusion, proteins were eluted with 2.5 mL 10 mmol/L gluthatione in PBS. Eluate was passed through PD-10 column (GE Healthcare, Uppsala, Sweden) to remove free gluthatione. To prepare Gst-tag-free proteins, we performed overnight cleavage at 4°C with PreScission protease (GE Healthcare, Uppsala, Sweden) followed by passage through Gst-trap column to remove free Gst protein. Protein-containing fractions were collected, checked with SDS-PAGE and kept at À20°C.

| Dot-blot experiments using C90-HRP
Dot-blot experiments were performed with Gst-tag-free proteins applied to the nitrocellulose membrane. After blocking of membrane with 3% BSA in TBS-T buffer for 1 hour and subsequent wash with TBS-T, C90-HRP (1 9 500 times dilution) was applied and incubated for 1 hour. After subsequent washing (3 times 10 minutes, TBS-T), membrane was dried and ECL signals were quantified using a digital Fujifilm LAS3000 imager and LAS3000 software. In competition, experiments increasing concentrations of test molecules were coincubated with C90-HRP conjugates. Experiments were performed in triplicates; data are presented as mean AE SEM. P < .05 was considered to be statistically significant.

| Cell culture, cell fractionation and coimmunoprecipitation experiments
SH-SY5Y human neuroblastoma cells were cultured in DMEM supplemented with 10% FBS in 5% CO 2 , 95% air at 37°C. Cell fractionation was performed as described in Ref. [5]. Immunoprecipitation

| SDS-PAGE and Western blot analysis
Protein (25 lg) was mixed with 29 SDS sample buffer, boiled for 5 minutes and loaded onto 4%-12% Bis-Tris precast gels (ThermoScientific, Rockford IL, USA). The samples were electrophoresed and transferred to the nitrocellulose membrane (Whatman, Maidstone, UK), and proteins of interest were detected with specific antibodies using SuperSignal West Pico enhanced chemiluminescence system (ThermoScientific, Rockford IL, USA). Western blot signals were analysed and quantified using a digital Fujifilm LAS3000 imager and LAS3000 software.

| Cytochrome oxidase activity assay
Cytochrome c (2.7 mg/mL) was reduced by incubation with DTT, and excess of reducing agent was removed by gel filtration on PD-10 column (GE Healthcare, Uppsala, Sweden). Cytochrome oxidase activity was measured by decrease in absorbance of ferrocytochrome c at 550 nm. Mouse brain mitochondria were isolated according to Ref. [4]. Isolated mitochondria from non-Tg mice, vehicle-treated 5xFAD mice and 5xFAD mice treated with GMP-1 were diluted in the buffer containing 10 mmol/L Tris-HCl, pH 7.0, 0.5 mol/L sucrose, 0,05% Triton X-100 to concentration 0.5 mg/mL.
After addition of ferrocytochrome c, latency in 550 nm absorbance was immediately measured. Each group included six mice, and experiments were performed in triplicate. Data are presented as mean AE SEM. P < .05 was considered to be statistically significant.

| Transgenic drosophila experiments
The fly lines containing single and double copies of a signal-peptide-Ab 42 transgene were generated as described in Ref. [24]. The fly line expressing in the neurons dimer Ab 42 peptide connected via a linker of 12 amino acids (T-Ab 42 ) was generated as described in Ref. [25].
Flies were maintained on the standard food containing 1% Agar, 8% Brewer's yeast, 8% fructose, 5% potato dry powder, 0.05% Nipagin, 0.1% ascorbate in humidified atmosphere at 25°C. Indicated amounts of compounds were added directly to the food during solidification. The fly mobility assay represents the percentage of flies that able to cross the line at 8 cm from the bottom of test tube in 10 seconds. Six independent measurements for each group were performed. Data are presented as mean AE SEM. P < .05 was considered to be statistically significant. The survival assay in flies expressing tandem Ab 42 in the neurons was calculated as percentage of adult flies carrying the transgene, plain wings phenotype, to the total number of hatched flies with plain + curly wings phenotype.
Six independent measurements for each group were performed.
Data are presented as mean AE SEM. P < .05 was considered to be statistically significant.

| In vivo mice experiments
Experiments with control and 5xFAD, APP/presenilin-1 transgenic mice 26  Mice were trained and tested on 2 consecutive days. On the training day, mice received a foot shock (0.5 mA, 2 seconds) 5 seconds after being placed into the conditioning chamber. Thirty seconds afterwards, they were returned to their home cage again. Twenty-four hours after training, mice were tested by being returned to the conditioning chamber for 5 minutes without any shock, and freezing behaviour was recorded by the automated system and evaluated separately every minute. Freezing was defined as lack of movement except that required for respiration and is expressed as freezing time group values were calculated using the individual means. Data were tested for normality using a Kolmogorov-Smirnov test; differences between groups were calculated by one-way ANOVA followed by a Newman-Keuls post-hoc test, the alpha-error set to 0.05.    (Figure 2A and B). We  Figure 2C and D show that amount of mitochondria-associated APP is decreased by 50% in GMP-1-treated cells as compared to DMSO accompanied by increase in mature APP in P3 fraction.
We have previously shown that mitochondrial APP staining with antibodies against N-terminal epitope of APP molecule resulted in detection of several APP fragments of apparent molecular weight of 20-75 kD. 11 Results presented in Figure 2 suggest partial inhibition of APP association with mitochondria upon GMP-1 treatment and APP redistribution between different intracellular compartments. Drosophila flies are widely used in initial drug screening programmes as simple, fast and inexpensive in vivo model to assess drug toxicity and efficacy. Because of the lack of bloodbrain barrier, drosophila models allow to study CNS effect of drugs ingested from the food. We have used two previously described fly models expressing Ab 1-42 as well as more aggregation-prone and toxic tandem Ab 1-42 (T-Ab 1-42 ) in CNS. Transgenic drosophila expressing Ab 1-42 in CNS had reduced mobility in comparison with wild-type flies. 24 Figure 3A shows that the treatment of Ab 1-42 -expressing flies with 50 lmol/L of GMP-1 rescued motility defects associated with brain Ab 1-42 expression.

|
We have also used a T-Ab 1-42 drosophila model to assess the effect GMP-1 on toxicity of aggregation-prone Ab species. It has been previously shown that hatching of T-Ab 1-42 -expressing flies was compromised at 25°C in comparison with 18°C incubation temperatures. 25 We have observed that addition of GMP-1 into the food of T-Ab 1-42 -expressing flies growing at 25°C rescued hatching to the levels similar to 18°C ( Figure 3B).
These results suggest neuroprotective effect of GMP-1 in drosophila Ab 1-42 -expressing models.

Effect on behaviour, memory, amyloid beta accumulation, neuroinflammation and mitochondrial function
The 5xFAD model is a double transgenic APP/PS1 mouse model that coexpresses five AD mutations leading to accelerated plaque formation and increased Ab 1-42 production. 26 Amyloid deposition and gliosis begin at 2 months accompanied by robust neuronal loss starting from 3 months of age as well as cognitive and memory decline. 26 Moreover, Devi and Ohno have reported mitochondrial dysfunction in the brains of 5xFAD mice and found full-length APP as well APP C-terminal fragment C99 in isolated brain mitochondria fractions. 27 Four groups of 15 animals were used as follows: tg mice having 0.5% DMSO in their drinking water ad libitum as placebo; 5xFAD tg mice that received 16.7 mg/kg of compound in the drinking water from 3 weeks of age until the end of experiment at 6 months of age; tg mice that received 16.7 mg/kg of compound in the drinking water at the age of 5.5 months during 2 weeks; non-tg littermates receiving 0.5% DMSO in drinking water. Because of its physicochemical properties such as molecular mass of 229 Da, XLogP coefficient 1.67, small polar surface area GMP-1 was predicted to penetrate blood-brain barrier by several ADME predictors. Therefore, oral administration route was chosen and the dose corresponded to the 100 lmol/L of GMP-1 dissolved in water. Both acute and chronic treatments with GMP-1 were safe-no differences between different treatment groups were observed in Irwine general health test, body mass or internal organs evaluation (data not shown). Results of behaviour and memory tests are presented in F I G U R E 5 Effect of GMP-1 treatment on total APP and Ab as well as mitochondria-associated APP. A, Western blot of total brain extract using anti-APP (22c11) antibodies. B, Western blot of total brain extract using anti-Ab 1-42 antibodies. C, Quantification results of Western blot staining. Results (means AE SEM) are represented as percentages of signals measured in placebo-treated mice vs GMP-1-treated mice. *P < .05, n = 6 F I G U R E 6 Immunohistochemical assessment of GMP-1 treatment on amyloid plaques accumulation and neuroinflammation. A, Amyloid plaque area in cortex and hippocampus measured as anti-APP (6E10) antibody staining. Placebo-treated, GMP-1 chronically treated, GMP-1 acutely treated 5xFAD mice as well as non-transgenic animals indicated as A, B, C and D, respectively. Differences between groups were calculated by one-way ANOVA followed by a Newman-Keuls post-hoc test, the alpha-error set to 0.05. *P < .05, ***P < .001, n = 6. B, Representative images of brain slices from 6-month-old 5xFAD mice stained with 6E10 antibodies and Alexa Fluor â 488 secondary antibodies. Arrows indicate area of cortex (C) and hippocampus (H). C, Astrocytosis in the cortex and hippocampus measured as anti-GFAP antibody staining. Placebo-treated, GMP-1 chronically treated, GMP-1 acutely treated 5xFAD mice as well as non-transgenic animals indicated as A, B, C and D, respectively. Differences between groups were calculated by one-way ANOVA followed by a Newman-Keuls post-hoc test, the alpha-error set to 0.05. *P < .05, **P < .01, n = 6. D. Microglia activation in the cortex and hippocampus measured as anti-CD11b antibody staining. Placebo-treated, GMP-1 chronically treated, GMP-1 acutely treated 5xFAD mice as well as non-transgenic animals indicated as A, B, C and D, respectively. Differences between groups were calculated by one-way ANOVA followed by a Newman-Keuls post-hoc test, the alpha-error set to 0.05. **P < .01, n = 6 littermates ( Figure 4A). Next, we tested 5xFAD mice with hippocampus-dependent contextual fear conditioning, in which mice learn to associate a distinct context with an aversive foot shock. Wild-type mice exhibited a robust conditioned fear response as assessed by freezing (the absence of all but respiratory movements) when placed back into the conditioning chamber 1 day after training. Both acute and chronically GMP-1-treated 5xFAD mice exhibited higher levels of freezing as compared to untreated animals especially when PAVLOV ET AL.
| 3471 calculating freezing time at minutes 4 and 5 ( Figure 4B). These results indicated restorative effect of GMP-1 treatment on hippocampus-dependent memory formation. To understand underlying mechanism of GMP-1 effect on mice memory and behaviour, we have performed analysis of brain APP and Ab content as well as immunohistochemical analysis of inflammatory markers and mitochondrial function. Analysis of total APP in the brain homogenate showed no differences between GMP-1-and placebo-treated groups, whereas Ab 1-42 levels were significantly lower in the GMP-1-treated group ( Figure 5). The levels of APP associated with brainisolated mitochondria, albeit lower in the GMP-1-treated group, did not reach statistical significance ( Figure 5C). In two brain regions, cortex and hippocampus, we immunohistochemically assessed amyloid beta plaque load as well as astrocytosis and microglia activation.
Results presented in Figure 6A indicate that the Ab plaque load in the cortex and hippocampus decreased in the 5xFAD group chronically treated with GMP-1 in comparison with placebo-treated and acutely treated GMP-1 group. Figure 6B shows representative images of brain sections containing cortex and hippocampus stained with 6E10 antibodies. In brain tissue of 6-month-old 5xFAD mice, 6E10 antibodies recognized predominantly plaque pathology despite the fact that they can react with full-length APP. GMP-1 treatment significantly reduced number of plaques and antibody-stained area both in the cortex and hippocampus as compared with placebo-treated animals. The level of astrocytosis measured as GFAP staining was significantly decreased in hippocampal sections of 5xFAD group chronically treated with GMP-1 in comparison with placebo-treated and acutely treated GMP-1 group ( Figure 6C). Microglia activation levels measured as CD11b staining were significantly reduced in the cortex of acutely and chronically GMP-1-treated 5xFAD groups as compared to placebo-treated 5xFAD mice ( Figure 6D). Finally, we assessed brain mitochondrial function by measurement of cytochrome c oxidase activity of isolated brain mitochondria. We found that cytochrome c oxidase activity is significantly increased in GMP-1-treated 5xFAD mice in comparison with placebo-treated group ( Figure 7).

| DISCUSSION
Several studies link mitochondrial dysfunction in neurodegenerative disorders with abnormal accumulation of disease-associated proteins in mitochondria. [2][3][4][5][6] In this view, targeting intracellular protein trafficking and preventing abnormal accumulation of disease-related proteins in mitochondria hold a promise as a new therapeutic strategy for various neurodegenerative disorders. 28 Molecular chaperones Hsp70 and Hsp90 regulate variety of cellular processes including general protein folding, degradation and trafficking. 29 They have been described as potential targets for the treatment of neurodegenerative disorders; however, direct activation or inhibition of Hsp70/Hsp90 could lead to undesirable side-effects especially in lifelong treatment scenario. The action of molecular chaperones Hsp70/Hsp90 is mediated by the cochaperones, proteins interacting with molecular chaperones and providing their functional specificity. 30 Disruption of specific protein-protein interactions within molecular chaperone network would not elicit major disturbance in molecular chaperone system. Here, we described development of inhibitors of molecular interaction between Hsp70/ Hsp90 and Tom70 a mitochondrial protein import receptor and TPR domain co-chaperone. We have used in silico docking to identify molecules that have capacity to interact with TPR domain of Tom70 and inhibit its interaction with the Hsp70/Hsp90. The identified compound, 2-(methoxymethyl)pyrimido[1,2-a]benzimidazol-4-ol, GMP-1, was found to compete with Hsp90-derived peptide for Tom70 binding. It has to be mentioned that GMP-1 interaction with TPR domains has relatively low affinity for Tom70 as well as relatively low specificity towards other TPR proteins such as PP5, FKBP51 and Tom34.
We are currently executing hit-to-lead programme to identify compounds with improved affinity/specificity as well as their drug-likeness. GMP-1 analogues have previously been patented and described as CNS drugs that have depressing and ataractic effect and could be used to relieve anxiety and tension states (patent US4109087 A, 1978). They also reduced local inflammation measured by carageenan oedema assay and delayed hypersensitivity test in rats (patent US4109087 A, 1978). It is well known that progressive Ab accumulation in the brain triggers neuronal hyperexcitation both in AD animal models and patients with AD often resulting in seizures 31,32 ; therefore, compounds inhibiting such overexcitation could have neuroprotective effect. Our results in 5xFAD mice open field test indicate that GMP-1 could relieve anxiety state to the levels seen in wild-type animals. Also GMP-1-treated animals had reduced levels of astrocytosis and microglia activation in comparison with placebo-treated mice indicating reduced neuroinflammation. It is not currently known whether the reduced brain inflammation is linked to reduced brain Ab load or it exerts an independent effect on astrocytes and microglial cells. We have unexpectedly observed reduced levels of total Ab and amyloid plaques upon GMP-1 treatment. One plausible explanation would suggest that modulation of molecular chaperone network can also modify molecular chaperone involvement into intracellular Ab degradation.
With regard to mitochondrial metabolism, we have observed restoration of cytochrome oxidase activity in the GMP-1-treated mice. We have also found reduced levels of mitochondria-associated APP in the F I G U R E 7 Cytochrome oxidase activity measurement in isolated brain mitochondria. Result bars represent specific activity (means AE SEM) of cytochrome oxidase measured as described in Materials and Methods. *P < .05, n = 6 GMP-1-treated mice; however, because of the relatively low number of studied animals, the values did not reach statistical significance.
Finally, we have proposed a new treatment strategy for AD protecting mitochondrial function via modulation of molecular chaperone network. GMP-1 exhibits neuroprotective effect in drosophila and mice AD models resulting in increased fly viability and memory improvement in 5xFAD mice.