Subacute Ingestion of Caffeine and Oolong Tea Increases Fat Oxidation without Affecting Energy Expenditure and Sleep Architecture: A Randomized, Placebo-Controlled, Double-Blinded Cross-Over Trial.

Ingesting oolong tea or caffeine acutely increases energy expenditure, and oolong tea, but not caffeine, stimulates fat oxidation. The acute effects of caffeine, such as increased heart rate and interference with sleep, diminish over 1-4 days, known as caffeine tolerance. During each 14-day session of the present study, 12 non-obese males consumed oolong tea (100 mg caffeine, 21.4 mg gallic acid, 97 mg catechins and 125 mg polymerized polyphenol), caffeine (100 mg), or placebo at breakfast and lunch. On day 14 of each session, 24-h indirect calorimetry and polysomnographic sleep recording were performed. Caffeine and oolong tea increased fat oxidation by ~20% without affecting energy expenditure over 24-h. The decrease in the respiratory quotient by oolong tea was greater than that by caffeine during sleep. The effect of oolong tea on fat oxidation was salient in the post-absorptive state. These findings suggest a role of unidentified ingredients in oolong tea to stimulate fat oxidation, and this effect is partially suppressed in a postprandial state. Two weeks of caffeine or oolong tea ingestion increased fat oxidation without interfering with sleep. The effects of subacute ingestion of caffeine and oolong tea differed from the acute effects, which is a particularly important consideration regarding habitual tea consumption.

Author Correction: Tau binding protein CAPON induces tau aggregation and neurodegeneration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Tau binding protein CAPON induces tau aggregation and neurodegeneration.

To understand the molecular processes that link Aß amyloidosis, tauopathy and neurodegeneration, we screened for tau-interacting proteins by immunoprecipitation/LC-MS. We identified the carboxy-terminal PDZ ligand of nNOS (CAPON) as a novel tau-binding protein. CAPON is an adaptor protein of neuronal nitric oxide synthase (nNOS), and activated by the N-methyl-D-aspartate receptor. We observed accumulation of CAPON in the hippocampal pyramidal cell layer in the AppNL-G-F -knock-in (KI) brain. To investigate the effect of CAPON accumulation on Alzheimer's disease (AD) pathogenesis, CAPON was overexpressed in the brain of AppNL-G-F mice crossbred with MAPT (human tau)-KI mice. This produced significant hippocampal atrophy and caspase3-dependent neuronal cell death in the CAPON-expressing hippocampus, suggesting that CAPON accumulation increases neurodegeneration. CAPON expression also induced significantly higher levels of phosphorylated, oligomerized and insoluble tau. In contrast, CAPON deficiency ameliorated the AD-related pathological phenotypes in tauopathy model. These findings suggest that CAPON could be a druggable AD target.

Brief internet-based cognitive behavior therapy program with a supplement drink improved anxiety and somatic symptoms in Japanese workers.

BACKGROUND: Self-help cognitive behavior therapy (CBT) is a useful approach for the treatment of psychological problems. Recent research on the effectiveness of self-help internet-based CBT (ICBT) indicates that the paradigm moderately improves psychological problems. Furthermore, previous studies have shown that food and drinks containing supplements improve various health conditions. We investigated the effect of a brief self-help ICBT administered with a supplement drink on psychological well-being and somatic symptoms. METHODS: In total, 101 healthy workers were enrolled in the 4-week ICBT program, which consisted of psychoeducation on stress management, behavior activation, and cognitive restructuring. The supplement soft drink was taken every day during the program. The participants were instructed to watch on-demand video clips and read the self-help guidebook and supporting comic strip weekly on the Internet or smartphone. The Japanese version of the Profile of Mood States (POMS) was administered before and after completion of the program. Scores on the POMS tension-anxiety (POMS-TA), depression (POMS-D), and fatigue (POMS-F) subscales were used to assess the effect of the program. Somatic symptoms were assessed using the Brief Job Stress Questionnaire. RESULTS: In total, 75 participants continued the program for 4 weeks; however, of those, 27 failed to complete all weekly tasks or meet the post-assessment deadlines. Therefore, the data of 48 participants were included in the analysis. Pre-post intervention comparisons using paired t-tests revealed significant improvement on the POMS-TA, but not the POMS-D or POMS-F subscales. Moreover, participants reported a significant reduction in the severity of low back pain. CONCLUSION: Our brief intervention moderately improved anxiety levels and the symptom of low back pain. These findings suggest that the brief ICBT program is effective in non-patient populations. Future directions for brief ICBT are discussed. TRIAL REGISTRATION: This study was registered on February 10, 2016 at UMIN. The registration number is UMIN000020962.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

OBJECTIVE: Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. APPROACH AND RESULTS: To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Involvement of calpains in adult neurogenesis: implications for stroke.

Calpains are ubiquitous proteases involved in cell proliferation, adhesion and motility. In the brain, calpains have been associated with neuronal damage in both acute and neurodegenerative disorders, but their physiological function in the nervous system remains elusive. During brain ischemia, there is a large increase in the levels of intracellular calcium, leading to the activation of calpains. Inhibition of these proteases has been shown to reduce neuronal death in a variety of stroke models. On the other hand, after stroke, neural stem cells (NSC) increase their proliferation and newly formed neuroblasts migrate towards the site of injury. However, the process of forming new neurons after injury is not efficient and finding ways to improve it may help with recovery after lesion. Understanding the role of calpains in the process of neurogenesis may therefore open a new window for the treatment of stroke. We investigated the involvement of calpains in NSC proliferation and neuroblast migration in two highly neurogenic regions in the mouse brain, the dentate gyrus (DG) and the subventricular zone (SVZ). We used mice that lack calpastatin, the endogenous calpain inhibitor, and calpains were also modulated directly, using calpeptin, a pharmacological calpain inhibitor. Calpastatin deletion impaired both NSC proliferation and neuroblast migration. Calpain inhibition increased NSC proliferation, migration speed and migration distance in cells from the SVZ. Overall, our work suggests that calpains are important for neurogenesis and encourages further research on their neurogenic role. Prospective therapies targeting calpain activity may improve the formation of new neurons following stroke, in addition to affording neuroprotection.

Calpain-dependent cleavage of N-cadherin is involved in the progression of post-myocardial infarction remodeling.

Enzymatic proteolysis by calpains, Ca(2+)-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast(-/-) mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast(+/+) mice. The activity of cardiac calpains in Cast(+/+) mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast(-/-) mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast(-/-) and Cast(+/+) mice, Cast(-/-) mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast(+/+) mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast(-/-) mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast(+/+) mice. In the border zone of Cast(-/-) mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of ß-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.

Single App knock-in mouse models of Alzheimer's disease.

Experimental studies of Alzheimer's disease have largely depended on transgenic mice overexpressing amyloid precursor protein (APP). These mice, however, suffer from artificial phenotypes because, in addition to amyloid ß peptide (Aß), they overproduce other APP fragments. We generated knock-in mice that harbor Swedish and Beyreuther/Iberian mutations with and without the Arctic mutation in the APP gene. The mice showed typical Aß pathology, neuroinflammation and memory impairment in an age-dependent manner.

Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD.

Calpastatin prevents NF-κB-mediated hyperactivation of macrophages and attenuates colitis.

Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated, reconstituted with either CAST-knockout (KO) or wild-type (WT) bone marrow, and subjected to dextran sulfate sodium-induced colitis. CAST-KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared with WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNF-α, and IFN-γ mRNA levels compared with WT controls. Mechanistic investigations using small interfering RNA and KO bone marrow to generate CAST-deficient macrophages showed that CAST deficiency during activation with bacterial pathogen associated molecular patterns, including heat-killed Enterococcus faecalis or CpG DNA, led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNF-α secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.

Loss of DARPP-32 and calbindin in multiple system atrophy.

We evaluated the immunohistochemical intensities of α-synuclein, phosphorylated α-synuclein (p-syn), dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), calbindin-D 28k, calpain-cleaved carboxy-terminal 150-kDa spectrin fragment, and tyrosine hydroxylase in multiple system atrophy (MSA). The caudate head, anterior putamen, posterior putamen, substantia nigra, pontine nucleus, and cerebellar cortex from six MSA brains, six age-matched disease control brains (amyotrophic lateral sclerosis), and five control brains were processed for immunostaining by standard methods. Immunostaining for α-synuclein, p-syn, or both was increased in all areas examined in oligodendrocytes in MSA. Immunostaining for DARPP-32 and calbindin-D 28k was most prominently decreased in the posterior putamen, where neuronal loss was most prominent. Immunostaining for DARPP-32 and calbindin-D 28k was also diminished in the anterior putamen and caudate head, where neuronal loss was less prominent or absent. Calbindin immunostaining was also decreased in the dorsal tier of the substantia nigra and cerebellar cortex. Loss of immunostaining for DARPP-32 and calbindin-D 28k compared with that of neurons indicates calcium toxicity and disturbance of the phosphorylated state of proteins as relatively early events in the pathogenesis of MSA.

Calpain-mediated ataxin-3 cleavage in the molecular pathogenesis of spinocerebellar ataxia type 3 (SCA3).

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology.

Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 co-localize in the motor neurons of amyotrophic lateral sclerosis patients, but how they are linked is not clear. Here we demonstrate that activation of calpain, a Ca2+-dependent cysteine protease, by upregulation of Ca2+-permeable AMPA receptors generates carboxy-terminal-cleaved TDP-43 fragments and causes mislocalization of TDP-43 in the motor neurons expressing glutamine/arginine site-unedited GluA2 of conditional ADAR2 knockout (AR2) mice that mimic the amyotrophic lateral sclerosis pathology. These abnormalities are inhibited in the AR2res mice that express Ca2+-impermeable AMPA receptors in the absence of ADAR2 and in the calpastatin transgenic mice, but are exaggerated in the calpastatin knockout mice. Additional demonstration of calpain-dependent TDP43 fragments in the spinal cord and brain of amyotrophic lateral sclerosis patients, and high vulnerability of amyotrophic lateral sclerosis-linked mutant TDP43 to cleavage by calpain support the crucial role of the calpain-dependent cleavage of TDP43 in the amyotrophic lateral sclerosis pathology.

Metabolic stress response implicated in diabetic retinopathy: the role of calpain, and the therapeutic impact of calpain inhibitor.

To describe how a high fat diet (HFD) and hyperglycemia initiate a sequence of calpain activation and oxidative stress associated with neuro-degenerative changes in diabetic retinopathy (DR), hyperglycemia was induced with streptozotocin in mice lacking the gene for calpastatin (CAST KO), and in mice lacking the gene for the transcription factor NF-E2 related factor 2 (Nrf2 KO). All animals were fed a HFD. Retinal ganglion cell (RGC) density was estimated by labeling with fluorogold and immunohistochemistry. A potent calpain inhibitor, SNJ-1945, was administered daily until the animals were sacrificed. In vitro, oxidative stress-induced RGC loss was evaluated in a high glucose culture medium with and without SNJ-1945. Retinal mRNA of calpain-1 and calpain-2 was measured by quantitative RT-PCR. Pre-apoptotic substrates of cleaved α-fodrin and synaptophysin protein were quantified by immunoblot analysis. Axonal damage was examined in transverse sections of the optic nerve. A HFD and hyperglycemia significantly increased RGC and axonal degeneration 3 weeks into the experiment. Levels of cleaved α-fodrin were increased. In the CAST KO mice, the neurotoxicity was augmented significantly. Gene manipulation of CAST and orally administered SNJ-1945 successfully modified calpain levels in the retina and prevented RGC death. In vitro, a high-glucose culture of retinal cells without antioxidants showed more RGC death than that with antioxidant treatment. The expression of synaptophysin was significantly suppressed by SNJ-1945 treatment. These results suggest that calpain plays a crucial role in metabolic-induced RGC degeneration caused by hyperglycemia and oxidative stress. Antioxidant and calpain inhibition offers important opportunities for future neuroprotective treatment against RGC death in various metabolic stress-induced diseases including DR.

Calpastatin overexpression limits calpain-mediated proteolysis and behavioral deficits following traumatic brain injury.

Traumatic brain injury (TBI) results in abrupt, initial cell damage leading to delayed neuronal death. The calcium-activated proteases, calpains, are known to contribute to this secondary neurodegenerative cascade. Although the specific inhibitor of calpains, calpastatin, is present within neurons, normal levels of calpastatin are unable to fully prevent the damaging proteolytic activity of calpains after injury. In this study, increased calpastatin expression was achieved using transgenic mice that overexpress the human calpastatin (hCAST) construct under control of a calcium-calmodulin-dependent kinase II α promoter. Naïve hCAST transgenic mice exhibited enhanced neuronal calpastatin expression and significantly reduced protease activity. Acute calpain-mediated spectrin proteolysis in the cortex and hippocampus induced by controlled cortical impact brain injury was significantly attenuated in calpastatin overexpressing mice. Aspects of posttraumatic motor and cognitive behavioral deficits were also lessened in hCAST transgenic mice compared to their wildtype littermates. However, volumetric analyses of neocortical contusion revealed no histological neuroprotection at either acute or long-term time points. Partial hippocampal neuroprotection observed at a moderate injury severity was lost after severe TBI. This study underscores the effectiveness of calpastatin overexpression in reducing calpain-mediated proteolysis and behavioral impairment after TBI, supporting the therapeutic potential for calpain inhibition. In addition, the reduction in spectrin proteolysis without accompanied neocortical neuroprotection suggests the involvement of other factors that are critical for neuronal survival after contusion brain injury.

Mechanistic involvement of the calpain-calpastatin system in Alzheimer neuropathology.

The mechanism by which amyloid-ß peptide (Aß) accumulation causes neurodegeneration in Alzheimer's disease (AD) remains unresolved. Given that Aß perturbs calcium homeostasis in neurons, we investigated the possible involvement of calpain, a calcium-activated neutral protease. We first demonstrated close postsynaptic association of calpain activation with Aß plaque formation in brains from both patients with AD and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). Using a viral vector-based tracer, we then showed that axonal termini were dynamically misdirected to calpain activation-positive Aß plaques. Consistently, cerebrospinal fluid from patients with AD contained a higher level of calpain-cleaved spectrin than that of controls. Genetic deficiency of calpastatin (CS), a calpain-specific inhibitor protein, augmented Aß amyloidosis, tau phosphorylation, microgliosis, and somatodendritic dystrophy, and increased mortality in APP-Tg mice. In contrast, brain-specific CS overexpression had the opposite effect. These findings implicate that calpain activation plays a pivotal role in the Aß-triggered pathological cascade, highlighting a target for pharmacological intervention in the treatment of AD.

Critical role of calpain in axonal damage-induced retinal ganglion cell death.

Calpain, an intracellular cysteine protease, has been widely reported to be involved in neuronal cell death. The purpose of this study is to investigate the role of calpain activation in axonal damage-induced retinal ganglion cell (RGC) death. Twelve-week-old male calpstatin (an endogenous calpain inhibitor) knockout mice (CAST KO) and wild-type (WT) mice were used in this study. Axonal damage was induced by optic nerve crush (NC) or tubulin destruction induced by leaving a gelatin sponge soaked with vinblastine (VB), a microtubule disassembly chemical, around the optic nerve. Calpain activation was assessed by immunoblot analysis, which indirectly quantified the cleaved α-fodrin, a substrate of calpain. RGCs were retrogradely labeled by injecting a fluorescent tracer, Fluoro-Gold (FG), and the retinas were harvested and flat-mounted retinas prepared. The densities of FG-labeled RGCs harvested from the WT and CAST KO groups were assessed and compared. Additionally, a calpain inhibitor (SNJ-1945, 100 mg/kg/day) was administered orally, and the density of surviving RGCs was compared with that of the vehicle control group. The mean density of surviving RGCs in the CAST KO group was significantly lower than that observed in the WT group, both in NC and in VB. The mean density of surviving RGCs in the SNJ-1945-treated group was significantly higher than that of the control group. The calpain inhibitor SNJ-1945 has a neuroprotective effect against axonal damage-induced RGC death. This pathway may be an important therapeutic target for preventing this axonal damage-induced RGC death, including glaucoma and diabetic optic neuropathy and other CNS diseases that share a common etiology.

Potent amyloidogenicity and pathogenicity of Aß43.

The amyloid-ß peptide Aß42 is known to be a primary amyloidogenic and pathogenic agent in Alzheimer's disease. However, the role of Aß43, which is found just as frequently in the brains of affected individuals, remains unresolved. We generated knock-in mice containing a pathogenic presenilin-1 R278I mutation that causes overproduction of Aß43. Homozygosity was embryonic lethal, indicating that the mutation involves a loss of function. Crossing amyloid precursor protein transgenic mice with heterozygous mutant mice resulted in elevated Aß43, impairment of short-term memory and acceleration of amyloid-ß pathology, which accompanied pronounced accumulation of Aß43 in plaque cores similar in biochemical composition to those observed in the brains of affected individuals. Consistently, Aß43 showed a higher propensity to aggregate and was more neurotoxic than Aß42. Other pathogenic presenilin mutations also caused overproduction of Aß43 in a manner correlating with Aß42 and with the age of disease onset. These findings indicate that Aß43, an overlooked species, is potently amyloidogenic, neurotoxic and abundant in vivo.