Alzheimer's disease (AD) is the most common form of dementia associated with age. Neuropathologically, AD is characterized by the presence of extracellular amyloid deposits and intracellular neurofibrillary tangles, composed of aggregated β-amyloid (Aβ) peptides and hyperphosphorylated and aggregated tau protein, respectively, in the brain parenchyma. Such characteristics are believed to be tightly linked to additional pathological processes, including chronic neuroinflammation and neurodegeneration.
1- Querfurth H.W.
- LaFerla F.M.
Alzheimer's disease.
At the cellular level, various signaling cascades are aberrantly activated, which negatively influences cell function and survival. Of these, calpain overactivation has been investigated in AD because it arises early in the disease process.
2- Saito K.
- Elce J.S.
- Hamos J.E.
- Nixon R.A.
Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration.
Calpains are calcium-dependent cysteine proteases that are ubiquitously expressed as two isoforms, μ-calpain and m-calpain. Under physiological conditions, limited activation of calpain results in modification or activation of protein receptors, enzymes, transcription factors, and cytoskeletal proteins.
3Calcium-activated neutral proteinases as regulators of cellular function: implications for Alzheimer's disease pathogenesis.
, 4- Melloni E.
- Salamino F.
- Sparatore B.
The calpain-calpastatin system in mammalian cells: properties and possible functions.
, 5- Goll D.E.
- Thompson V.F.
- Taylor R.G.
- Zalewska T.
Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin.
, 6- Saido T.C.
- Sorimachi H.
- Suzuki K.
Calpain: new perspectives in molecular diversity and physiological-pathological involvement.
Notably, physiological activation of calpain has been implicated in key functions of developmental and adult synaptic plasticity.
7- Zadran S.
- Bi X.
- Baudry M.
Regulation of calpain-2 in neurons: implications for synaptic plasticity.
On the other hand, calpain overactivation, triggered by abnormally high calcium levels during pathological insults, can be a harmful force within the cell. In the central nervous system, exacerbated calpain activation has been linked to calcium-mediated cell injury found in ischemic stroke, spinal cord injury, traumatic brain injury, and wallerian degeneration.
8- Kamakura K.
- Ishiura S.
- Sugita H.
- Toyokura Y.
Identification of Ca2+-activated neutral protease in the peripheral nerve and its effects on neurofilament degeneration.
, 9- Bartus R.T.
- Elliott P.J.
- Hayward N.J.
- Dean R.L.
- Harbeson S.
- Straub J.A.
- Li Z.
- Powers J.C.
Calpain as a novel target for treating acute neurodegenerative disorders.
, 10- Li Z.
- Hogan E.L.
- Banik N.L.
Role of calpain in spinal cord injury: increased mcalpain immunoreactivity in spinal cord after compression injury in the rat.
, 11- Saatman K.E.
- Bozyczko-Coyne D.
- Marcy V.
- Siman R.
- McIntosh T.K.
Prolonged calpain-mediated spectrin breakdown occurs regionally following experimental brain injury in the rat.
Regarding the AD brain, calpains have regulated the proteolytic processing of the amyloid precursor protein (APP) and phosphorylation and proteolysis of tau, leading to a hypothesis that selective calpain inhibitors are potential therapeutic strategies for AD.
12- Siman R.
- Card J.P.
- Davis L.G.
Proteolytic processing of beta-amyloid precursor by calpain I.
, 13- Klafki H.
- Abramowski D.
- Swoboda R.
- Paganetti P.A.
- Staufenbiel M.
The carboxyl termini of beta-amyloid peptides 1-40 and 1-42 are generated by distinct gamma-secretase activities.
, 14- Grynspan F.
- Griffin W.R.
- Cataldo A.
- Katayama S.
- Nixon R.A.
Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease.
, 15- Mathews P.M.
- Jiang Y.
- Schmidt S.D.
- Grbovic O.M.
- Mercken M.
- Nixon R.A.
Calpain activity regulates the cell surface distribution of amyloid precursor protein: inhibition of calpains enhances endosomal generation of beta-cleaved C-terminal APP fragments.
, 16- Liang B.
- Duan B.Y.
- Zhou X.P.
- Gong J.X.
- Luo Z.G.
Calpain activation promotes BACE1 expression, amyloid precursor protein processing, and amyloid plaque formation in a transgenic mouse model of Alzheimer disease.
However, the potential use of known calpain inhibitors as therapeutic tools in AD is limited because of their low cellular penetration, poor selectivity, and kinetics. More recently, A-705253, a novel calpain inhibitor capable of inhibiting calpain in nanomolar concentrations and with enhanced oral bioavailability, water solubility, and metabolic stability, has been characterized and may be a promising candidate of a therapeutic drug for AD.
17- Lubisch W.
- Beckenbach E.
- Bopp S.
- Hofmann H.P.
- Kartal A.
- Kastel C.
- Lindner T.
- Metz-Garrecht M.
- Reeb J.
- Regner F.
- Vierling M.
- Moller A.
Benzoylalanine-derived ketoamides carrying vinylbenzyl amino residues: discovery of potent water-soluble calpain inhibitors with oral bioavailability.
In this study, we sought to preclinically assess the therapeutic effects of A-705253 on aged 3xTgAD mice and to determine the possible molecular mechanisms involved in its actions.
Materials and Methods
Animal Treatments
3xTgAD mice harboring a presenilin1 mutation (PS1
M146V), the Swedish double mutation in APP (APP
KM670/671ML), and a frontotemporal dementia mutation in tau (tau
P301L) were used for all experiments.
18- Oddo S.
- Caccamo A.
- Shepherd J.D.
- Murphy M.P.
- Golde T.E.
- Kayed R.
- Metherate R.
- Mattson M.P.
- Akbari Y.
- LaFerla F.M.
Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction.
Strain-matched nontransgenic mice (nTg), 129/C57BL/6, were used as controls. Calpain inhibitor A-705253 [
N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl] benzamide] was dissolved in 2% sucrose solution and administered to the mice in their drinking water at a dose of either 40 or 80 mg/kg per day. A-705253 was kindly donated by Abbott Laboratories (Ludwigshafen, Germany). Mice were treated for 3 months, from the age of 15 months to the age of 18 months. No changes in total water consumption and body weight were found between vehicle and A-705253–treated mice. All procedures used in the present study followed the Principles of Laboratory Animal Care from NIH publication 85-23 and were approved by the University of California, Irvine, Institutional Animal Care and Use Committee.
Behavior Paradigms
Behavior paradigms to assess cognition were performed for all groups of mice between the ages of 17 and 18 months, as previously described.
19- Medeiros R.
- Kitazawa M.
- Caccamo A.
- Baglietto-Vargas D.
- Estrada-Hernandez T.
- Cribbs D.H.
- Fisher A.
- Laferla F.M.
Loss of muscarinic m(1) receptor exacerbates Alzheimer's disease-like pathology and cognitive decline.
Novel object recognition, Morris water maze, and contextual fear conditioning are briefly described herein.
Novel Object Recognition
Each mouse was habituated to an empty arena for 3 consecutive days. On the first day of testing, mice were exposed to two identical objects placed at opposite ends of the arena for 5 minutes. Twenty-four hours later, the mouse was returned to the test box, this time with one familiar object and one novel object. Time spent exploring the objects was recorded for 5 minutes. The recognition index represents the percentage of the time that mice spend exploring the novel object. Objects used in this task were carefully selected to prevent preference or phobic behavior.
Morris Water Maze
Mice were trained to swim to a circular clear Plexiglas platform submerged 1.5 cm beneath the surface. Four trials were performed per day, for 60 seconds each, with 5 minutes between trials. Mice were trained for as many days as needed for the group to reach the training criterion of 25 seconds. The probe test was assessed 24 hours after the last trial, with the platform removed. Performance was monitored with the EthoVision XT video-tracking system (Noldus Information Technology, Leesburg, VA).
Contextual Fear Conditioning
During training, mice were placed in the fear conditioning chamber and allowed to explore for 2 minutes before receiving three electric foot shocks (duration: 1 second; intensity, 0.2 mA; intershock interval, 2 minutes). Animals were returned to the home cage 30 seconds after the last foot shock. Twenty-four hours later, behavior in the conditioning chamber was videorecorded during 5 minutes and subsequently analyzed for freezing behavior.
Tissue Preparation
Mice were deeply anesthetized with sodium pentobarbital and sacrificed by perfusion transcardially with 0.1 mol/L PBS (pH 7.4) solution. The right brain hemispheres were fixed for 48 hours in 4% paraformaldehyde and cryoprotected in 30% sucrose for immunohistochemical (IHC) analysis. Frozen brains were divided coronally into sections (40 μm thick) using an SM2010R freezing microtome (Leica Microsystems, Bannockburn, IL), serially collected in cold 0.02% sodium azide, and stored at 4°C. The left hemispheres were snap frozen on dry ice and subjected to protein extraction sequentially using T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) and 70% formic acid. The supernatant was fractionated and stored at −80°C. Protein concentration in the supernatant was determined using the Bradford assay.
Immunoblotting
Equal protein amounts were separated on a 4% to 12% gradient SDS-PAGE, transferred to a nitrocellulose membrane, and incubated overnight at 4°C with primary antibody. The following primary antibodies were used in this study: phospho-glycogen synthetase kinase (GSK3β; Ser9), human APP-CT20, Disintegrin and metalloproteinase domain-containing protein (ADAM)10, ADAM17, BACE1, GSK3β, CDK5 (Calbiochem, San Diego, CA), human tau (HT7), phospho-tau AT8 (phospho-Ser202/Thr205), phospho-tau AT180 (phospho-Thr231), phospho-tau AT270 (phospho-Thr181) (Thermo Scientific), Aβ1–16 (6E10) (Covance Research Products, Denver, PA), protein phosphatase 2A (PP2A), p35, synaptophysin (Sigma-Aldrich, St. Louis, MO), neprilysin, insulin-degrading enzyme (IDE), ABCA1, apolipoprotein E, low density lipoprotein receptor-related protein (LRP), liver X receptor (LXR), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membranes were incubated with adjusted secondary antibodies coupled to horseradish peroxidase. The immunocomplexes were visualized using the SuperSignal West Pico Kit (Thermo Scientific). Band density measurements were made using ImageJ 1.36b imaging software (NIH, Bethesda, MD).
ELISA Data
For the determination of the Aβ levels, T-PER soluble fractions were loaded directly onto enzyme-linked immunosorbent assay (ELISA) plates, whereas the formic acid supernatants (insoluble fractions) were diluted 1:20 in a neutralization buffer (1 mol/L Tris base and 0.5 mol/L NaH2PO4) before loading. MaxiSorp immunoplates (Nunc, Rochester, NY) were coated with mAb20.1 antibody (a gift from Dr. William E. Van Nostrand, Stony Brook University, Stony Brook, NY) at a concentration of 25 μg/mL in coating buffer (0.1 mol/L Na2CO3, pH 9.6) and blocked with 3% bovine serum albumin. Standard solutions for both Aβ40 and Aβ42 were made in the antigen capture buffer (20 mmol/L NaH2PO4, 2 mmol/L EDTA, 0.4 mol/L NaCl, 0.05% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and 1% bovine serum albumin, pH 7.0) and loaded onto ELISA plates in duplicate. Samples were then loaded (also in duplicate) and incubated overnight at 4°C. Plates were then washed and probed with either horseradish peroxidase–conjugated anti-Aβ40 (C49) or anti-Aβ42 (D32) (a gift from Drs. Vitaly Vasilevko and David H. Cribbs, University of California, Irvine) overnight at 4°C. The chromogen was 3,3′,5,5′-tetramethylbenzidine, and the reaction was stopped by 30% phosphoric acid. The plates were read at 450 nm using a plate reader (Molecular Dynamics, Sunnyvale, CA). The readings were then normalized to protein concentrations of the samples.
IHC Data
Antigen retrieval was optimized using a 90% formic acid solution for 7 minutes for Aβ staining. Free-floating sections were pretreated with 3% hydrogen peroxide and 10% methanol in Tris-buffered saline (TBS) for 30 minutes to block endogenous peroxidase activity. After a TBS wash, sections were incubated once in 0.1% Triton X-100 in TBS for 15 minutes and once with 2% bovine serum albumin in 0.1% Triton X-100 in TBS for 30 minutes. Sections were then incubated overnight at 4°C with anti-Aβ42 (D32) (a gift from Drs. Vitaly Vasilevko and David H. Cribbs, University of California, Irvine) or anti-CD45 (AbD Serotec, Raleigh, NC) with 5% normal serum in TBS. After the appropriate biotinylated secondary antibody (1:200 in TBS + 2% bovine serum albumin + 5% normal serum), sections were processed using the Vectastain Elite ABC reagent and 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA), according to the manufacturer's instructions. Sections were then mounted on gelatin-coated slides, dehydrated in graded ethanol, cleared in xylene, and coverslipped with DPX mounting medium (BDH Laboratory Supplies, Poole, UK).
The immunostaining was assessed at six brain coronal levels. Specifically, six alternate sections (40 μm thick) of the brain with an individual distance of approximately 160 μm were obtained between 1.34 and 2.54 mm posterior to the bregma. Images of stained hippocampal, entorhinal cortex, and amygdale area were acquired using an Axiocam digital camera and AxioVision 4.6 software connected to an Axioskop 50 microscope (Carl Zeiss MicroImaging, Thornwood, NY). A threshold optical density that best discriminated staining from the background was obtained using the ImageJ 1.36b imaging software (NIH). All histological assessments were made by an examiner blinded to sample identities (R.M., M.K., and D.B.V.).
Immunofluorescence
Sections were first blocked with 3% normal serum, 2% bovine serum albumin, and 0.1% Triton X-100 in TBS for 1 hour at room temperature. By using the same buffer solution, sections were incubated overnight at 4°C with the following primary antibodies: anti-Aβ1–16 (6E10) (Covance Research Products), anti-postsynaptic density protein 95 (PSD-95), anti-glial fibrillary acidic protein (Millipore, Billerica, MA), human tau (HT7) (Thermo Scientific) (1:1000), anti-ionized calcium binding adaptor molecule 1 (Iba-1) (Wako Chemicals, Richmond, VA) and/or phospho-tau PHF-1 (phospho-S396/S404) (a gift from Dr. Peter Davies, Albert Einstein College of Medicine, Manhasset, NY). Sections were then rinsed and incubated for 1 hour with secondary Alexa Fluor–conjugated antibodies (Invitrogen, Carlsbad, CA) at room temperature. Finally, sections were mounted onto gelatin-coated slides in Fluoromount-G (Southern Biotech, Birmingham, AL) and examined under a Leica DM2500 confocal laser microscope using the Leica Application Suite Advanced Fluorescence software (Leica Microsystems). The immunofluorescence was assessed at the same brain coronal levels previously described. Confocal images were acquired by sequential scanning using a z-separation of 1 μm using the Leica Application Suite Advanced Fluorescence software (Leica Microsystems).
Thioflavin S Staining
Sections were incubated in 0.5% thioflavin S in 50% ethanol for 10 minutes, differentiated twice in 50% ethanol, and washed in PBS solution. Staining was visualized under a confocal microscope. Volumetric imaging and image measurements were made using Imaris software (Bitplane Inc., South Windsor, CT). The thioflavin S levels represent the average value obtained by the analysis of images of the hippocampus, entorhinal cortex, and amygdala.
Statistical Analysis
All data are expressed as mean ± SEM. The statistical evaluation of the results was performed using one- or two-way analysis of variance (ANOVA). After significant ANOVAs, multiple post hoc comparisons were performed using Bonferroni's test. Some data were analyzed using the unpaired t-test. The accepted level of significance for the tests was P < 0.05. All tests were performed using the Statistica software package (StatSoft Inc., Tulsa, OK).
Discussion
Herein, we provide functional and molecular preclinical evidence that the novel calpain inhibitor A-705253 mitigates the AD-like pathological characteristics in aged 3xTgAD mice. Inhibition of calpain by A-705253 restored the cognitive function and prevented the synaptic degeneration. Through a decrease in BACE1-mediated production and an increase in ABCA1-mediated clearance of Aβ, A-705253 promoted a decline in the number of fibrillar amyloid plaques in the brain parenchyma. In addition, the oral treatment with A-705253 reduced the hyperphosphorylation of tau through a CDK5-dependent mechanism.
Calpains are multifunctional enzymes unique in their capability to cleave many substrates and, thus, trigger a wide range of cellular responses. In the central nervous system, inappropriate activation of calpain stimulates neuronal death and disrupts learning and memory. Evidence has also established that neurites and synaptic terminals are highly loaded with activated calpain, which may account for early synaptic loss and neuritic dystrophy in the AD brain.
2- Saito K.
- Elce J.S.
- Hamos J.E.
- Nixon R.A.
Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration.
, 24A “protease activation cascade” in the pathogenesis of Alzheimer's disease.
, 25- Trinchese F.
- Fa M.
- Liu S.
- Zhang H.
- Hidalgo A.
- Schmidt S.D.
- Yamaguchi H.
- Yoshii N.
- Mathews P.M.
- Nixon R.A.
- Arancio O.
Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.
Indeed, calpain inhibitors impede the cleavage of p35 to p25 and the subsequent cell death induced by Aβ.
26- Patrick G.N.
- Zukerberg L.
- Nikolic M.
- de la Monte S.
- Dikkes P.
- Tsai L.H.
Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
, 27- Lee M.S.
- Kwon Y.T.
- Li M.
- Peng J.
- Friedlander R.M.
- Tsai L.H.
Neurotoxicity induces cleavage of p35 to p25 by calpain.
Moreover, inhibition of calpain activity reestablishes normal synaptic plasticity in neuronal cultures from APP/PS1 mice through an enhancement in cAMP response element-binding (CREB) phosphorylation.
25- Trinchese F.
- Fa M.
- Liu S.
- Zhang H.
- Hidalgo A.
- Schmidt S.D.
- Yamaguchi H.
- Yoshii N.
- Mathews P.M.
- Nixon R.A.
- Arancio O.
Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.
Corroborating these findings, the calpain inhibitor A-705253 prevents
N-methyl-
d-aspartate–induced neuronal death and Aβ-induced synaptic dysfunction in hippocampal slice cultures.
28- Nimmrich V.
- Reymann K.G.
- Strassburger M.
- Schoder U.H.
- Gross G.
- Hahn A.
- Schoemaker H.
- Wicke K.
- Moller A.
Inhibition of calpain prevents NMDA-induced cell death and beta-amyloid-induced synaptic dysfunction in hippocampal slice cultures.
Similarly, the administration of this compound to rats diminishes the loss of cholinergic neurons in the nucleus basalis magnocellularis and prevents the cognitive dysfunction induced by the injection of Aβ
42 oligomers.
29- Granic I.
- Nyakas C.
- Luiten P.G.
- Eisel U.L.
- Halmy L.G.
- Gross G.
- Schoemaker H.
- Moller A.
- Nimmrich V.
Calpain inhibition prevents amyloid-beta-induced neurodegeneration and associated behavioral dysfunction in rats.
Notably, our study demonstrates that long-term oral treatment with A-705253 markedly reduces both Aβ and tau pathological characteristics in the brains of aged 3xTgAD mice. Of great relevance, inhibition of calpain also restored synaptic proteins and learning and memory to the level of nTg mice, which reinforce the idea that calpain may be important in the regulation of normal cognitive functions.
7- Zadran S.
- Bi X.
- Baudry M.
Regulation of calpain-2 in neurons: implications for synaptic plasticity.
These data suggest that A-705253 is neuroprotective and has disease-modifying effects in mice.
Further biochemical analyses reveal that A-705253 treatment resulted in a reduction in BACE1 levels, the putative β-secretase involved in APP metabolism and the rate-limiting factor in Aβ generation.
30- Sinha S.
- Anderson J.P.
- Barbour R.
- Basi G.S.
- Caccavello R.
- Davis D.
- Doan M.
- Dovey H.F.
- Frigon N.
- Hong J.
- Jacobson-Croak K.
- Jewett N.
- Keim P.
- Knops J.
- Lieberburg I.
- Power M.
- Tan H.
- Tatsuno G.
- Tung J.
- Schenk D.
- Seubert P.
- Suomensaari S.M.
- Wang S.
- Walker D.
- Zhao J.
- McConlogue L.
- John V.
Purification and cloning of amyloid precursor protein beta-secretase from human brain.
, 31- Vassar R.
- Bennett B.D.
- Babu-Khan S.
- Kahn S.
- Mendiaz E.A.
- Denis P.
- Teplow D.B.
- Ross S.
- Amarante P.
- Loeloff R.
- Luo Y.
- Fisher S.
- Fuller J.
- Edenson S.
- Lile J.
- Jarosinski M.A.
- Biere A.L.
- Curran E.
- Burgess T.
- Louis J.C.
- Collins F.
- Treanor J.
- Rogers G.
- Citron M.
Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.
, 32- Yan R.
- Bienkowski M.J.
- Shuck M.E.
- Miao H.
- Tory M.C.
- Pauley A.M.
- Brashier J.R.
- Stratman N.C.
- Mathews W.R.
- Buhl A.E.
- Carter D.B.
- Tomasselli A.G.
- Parodi L.A.
- Heinrikson R.L.
- Gurney M.E.
Membrane-anchored aspartyl protease with Alzheimer's disease beta-secretase activity.
, 33- Cai H.
- Wang Y.
- McCarthy D.
- Wen H.
- Borchelt D.R.
- Price D.L.
- Wong P.C.
BACE1 is the major beta-secretase for generation of Abeta peptides by neurons.
Our findings are in agreement with previous evidence that has demonstrated that overexpression of m-calpain in APP695myc-expressing HEK293 cells augments the levels of BACE1 and APP CTFs. Likewise, Aβ-induced BACE1 up-regulation in cultured cortical neurons and Aβ deposition in APP/PS1 mice are attenuated by overexpression of endogenous calpain inhibitor calpastatin.
16- Liang B.
- Duan B.Y.
- Zhou X.P.
- Gong J.X.
- Luo Z.G.
Calpain activation promotes BACE1 expression, amyloid precursor protein processing, and amyloid plaque formation in a transgenic mouse model of Alzheimer disease.
More important, the calpain-mediated BACE1 induction and the consequent Aβ accumulation have been linked to the release of proteolytic fragment p25 from p35 and activation of CDK5.
34- Cruz J.C.
- Kim D.
- Moy L.Y.
- Dobbin M.M.
- Sun X.
- Bronson R.T.
- Tsai L.H.
p25/Cyclin-dependent kinase 5 induces production and intraneuronal accumulation of amyloid beta in vivo.
, 35- Wen Y.
- Yu W.H.
- Maloney B.
- Bailey J.
- Ma J.
- Marie I.
- Maurin T.
- Wang L.
- Figueroa H.
- Herman M.
- Krishnamurthy P.
- Liu L.
- Planel E.
- Lau L.F.
- Lahiri D.K.
- Duff K.
Transcriptional regulation of beta-secretase by p25/cdk5 leads to enhanced amyloidogenic processing.
A second possible mechanism by which A-705253 reduces Aβ levels in the brain is mediated by the increase in ABCA1 levels. ABCA1 is a member of the ATP-binding cassette family of active transporters that was recently linked to the clearance of Aβ in the AD brain. Although deletion of ABCA1 increases the Aβ deposition, its overexpression has an opposite effect through the increase in Aβ clearance, in the PDAPP mice.
22- Wahrle S.E.
- Jiang H.
- Parsadanian M.
- Hartman R.E.
- Bales K.R.
- Paul S.M.
- Holtzman D.M.
Deletion of Abca1 increases Abeta deposition in the PDAPP transgenic mouse model of Alzheimer disease.
, 23- Wahrle S.E.
- Jiang H.
- Parsadanian M.
- Kim J.
- Li A.
- Knoten A.
- Jain S.
- Hirsch-Reinshagen V.
- Wellington C.L.
- Bales K.R.
- Paul S.M.
- Holtzman D.M.
Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease.
ABCA1 expression is highly regulated, on both transcriptional and post-transcriptional levels. Given that ABCA1 contains a sequence rich in proline, glutamic acid, serine, and threonine, it is speculated that A-705253 increases ABCA1 levels in the 3xTgAD mouse brain by inhibiting its degradation by calpains.
36- Wang N.
- Chen W.
- Linsel-Nitschke P.
- Martinez L.O.
- Agerholm-Larsen B.
- Silver D.L.
- Tall A.R.
A PEST sequence in ABCA1 regulates degradation by calpain protease and stabilization of ABCA1 by apoA-I.
, 37- Chen W.
- Wang N.
- Tall A.R.
A PEST deletion mutant of ABCA1 shows impaired internalization and defective cholesterol efflux from late endosomes.
This hypothesis is supported by our data showing unchanged levels of LXR after the inhibition of calpain, because this is the main transcriptional factor associated with the up-regulation of ABCA1 expression.
38Transcriptional regulatory networks in lipid metabolism control ABCA1 expression.
Although we clearly demonstrate that inhibition of calpain reduces Aβ levels in the 3xTgAD mouse brain, others have shown a conflicting effect, in which inhibition of calpain activity, through the change in the intracellular distribution of APP toward the cell surface, increases both α- and β-cleavage and, consequently, Aβ levels.
15- Mathews P.M.
- Jiang Y.
- Schmidt S.D.
- Grbovic O.M.
- Mercken M.
- Nixon R.A.
Calpain activity regulates the cell surface distribution of amyloid precursor protein: inhibition of calpains enhances endosomal generation of beta-cleaved C-terminal APP fragments.
The reasons for such a discrepancy are unknown and may be the result of differences in the approaches applied. Additional studies are necessary to clarify the relationship between Aβ and calpain.
Dysregulation of kinase and/or phosphatase activity may lead to aberrant phosphorylation of tau in mature neurons, which, in turn, accumulates in the somatodendritic compartments and eventually develops into mature neurofibrillary tangles, resulting in neuronal degeneration and cognitive decline.
39- Grundke-Iqbal I.
- Iqbal K.
- Tung Y.C.
- Quinlan M.
- Wisniewski H.M.
- Binder L.I.
Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.
, 40- Sengupta A.
- Kabat J.
- Novak M.
- Wu Q.
- Grundke-Iqbal I.
- Iqbal K.
Phosphorylation of tau at both Thr 231 and Ser 262 is required for maximal inhibition of its binding to microtubules.
, 41- Schneider A.
- Biernat J.
- von Bergen M.
- Mandelkow E.
- Mandelkow E.M.
Phosphorylation that detaches tau protein from microtubules (Ser262, Ser214) also protects it against aggregation into Alzheimer paired helical filaments.
In this process, calpain displays an essential role by generating p25 from the cleavage of p35. Unlike p35, p25 is not readily degraded, and binding of p25 to CDK5 constitutively activates CDK5, causing hyperphosphorylation of tau, collapse of the cytoskeleton, and neuronal dystrophy and death.
26- Patrick G.N.
- Zukerberg L.
- Nikolic M.
- de la Monte S.
- Dikkes P.
- Tsai L.H.
Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration.
, 27- Lee M.S.
- Kwon Y.T.
- Li M.
- Peng J.
- Friedlander R.M.
- Tsai L.H.
Neurotoxicity induces cleavage of p35 to p25 by calpain.
Consistent with this idea, long-term oral treatment with A-705253 reduced the phosphorylation of tau at Ser202/Thr205 (AT8), Thr181 (AT270), Thr231/Ser235 (AT180), and Ser396/Ser404 (PHF-1) phosphorylated sites. Notably, such an effect was associated with a selective effect on p35 cleavage, because the brains of A-705253–treated 3xTgAD mice presented diminished levels of p25 fragment and unchanged levels of activated tau kinase GSK3β and tau phosphatase PP2A. More important, recent evidence has also reported that A-705253 prevents the cleavage of tau and the release of a cytotoxic 17-kDa fragment induced by Aβ
in vitro.
42- Sinjoanu R.C.
- Kleinschmidt S.
- Bitner R.S.
- Brioni J.D.
- Moeller A.
- Ferreira A.
The novel calpain inhibitor A-705253 potently inhibits oligomeric beta-amyloid-induced dynamin 1 and tau cleavage in hippocampal neurons.
Taken together, these data clearly indicate that A-705253 is highly effective in preventing calpain-mediated tau pathological characteristics.
Several lines of evidence have shown that microglia and astrocytes are attracted to Aβ deposits in which their overactivation elicits detrimental responses and can potentiate the disease progression in the AD brain.
43Inflammatory aspects of Alzheimer disease and other neurodegenerative disorders.
Herein, we demonstrate that A-705253 reduces the inflammatory response associated with Aβ accumulation. This reduction in gliosis may be either indirectly through the inhibition of AD-like pathological characteristics or directly by the modulation of glial cells. In fact, recent studies have shown that activation of calpain in microglia and astrocytes contributes to the pathological processes after experimental allergic encephalomyelitis and spinal cord injury in rats.
44- Shields D.C.
- Tyor W.R.
- Deibler G.E.
- Hogan E.L.
- Banik N.L.
Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis.
, 45- Shields D.C.
- Schaecher K.E.
- Hogan E.L.
- Banik N.L.
Calpain activity and expression increased in activated glial and inflammatory cells in penumbra of spinal cord injury lesion.
Moreover, the calpain inhibitor I exerts potent anti-inflammatory effects preventing the recruitment of neutrophils, expression of inducible nitric oxide synthase and cyclooxygenase-2 protein and activity, and, ultimately, the degree of peroxynitrite formation and tissue injury
in vivo.
46- Cuzzocrea S.
- McDonald M.C.
- Mazzon E.
- Siriwardena D.
- Serraino I.
- Dugo L.
- Britti D.
- Mazzullo G.
- Caputi A.P.
- Thiemermann C.
Calpain inhibitor I reduces the development of acute and chronic inflammation.
The relevance of calpain on acute and chronic inflammation has been associated with its capability to modulate the degradation of IκB in the proteasome, which is a fundamental step in the activation of transcription factor NF-κB.
47- Ruetten H.
- Thiemermann C.
Effect of calpain inhibitor I, an inhibitor of the proteolysis of I kappa B, on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat.
, 48- Virlos I.
- Mazzon E.
- Serraino I.
- Genovese T.
- Di Paola R.
- Thiemerman C.
- Siriwardena A.
- Cuzzocrea S.
Calpain I inhibitor ameliorates the indices of disease severity in a murine model of cerulein-induced acute pancreatitis.
Additional studies, however, are necessary to establish the role of calpain on NF-κB in the AD brain.
Overall, our study shows that the novel calpain inhibitor A-705253 reduces AD-like pathological characteristics in 3xTgAD mice. Because calpain overactivation contributes greatly to the enhancement of the neuropathological processes in the AD brain, the use of A-705253 could represent an attractive and clinically applicable therapeutic approach for AD.
Article info
Publication history
Published online: June 11, 2012
Accepted:
April 12,
2012
Copyright
© 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.