Expression and functional profiling of neprilysin, insulin-degrading enzyme, and endothelin-converting enzyme in prospectively studied elderly and Alzheimer's brain.

The brain steady state level of ß-amyloid (Aß) is determined by the balance between its production and removal, the latter through egress across blood and CSF barriers as well as Aß degradation. The major Aß-degrading enzymes are neprilysin (NEP), insulin-degrading enzyme (IDE), and endothelin-converting enzyme (ECE-1). Although evidence suggests that NEP is down-regulated in Alzheimer's disease (AD), the role of IDE and ECE in the Aß accumulation in aging and dementia remains less certain. In this study, we examined mRNA and protein expression, as well as biological activity of NEP, IDE, and ECE-1 in human frontal cortex by real-time RT-PCR for mRNA, immunoblotting for protein, and highly sensitive and specific fluorescence assays for activity. The relationships between Aß-degrading enzymes and pathologic measures and clinical features were also assessed. The results showed that NEP mRNA, protein level, and activity were decreased in AD compared with normal controls with no cognitive impairment (NCI). In contrast, IDE activity was unchanged, but there was higher expression of IDE mRNA, indicating a possible compensatory reaction because of deficits in activity. ECE-1 expression in AD brain showed no significant difference compared with age-matched controls. Correlation analyses suggested that NEP expression was correlated with Aß accumulation and clinical diagnosis, being lower in AD than in no cognitive impairment. In contrast, neither IDE nor ECE-1 correlated with Aß or clinical diagnosis. These findings provide additional support for NEP as the major protease involved in Aß degradation and suggest its possible therapeutic targeting in AD.

Effects of HNE-modification induced by Abeta on neprilysin expression and activity in SH-SY5Y cells.

The cerebral accumulation of beta-amyloid (Abeta) is a consistent feature of and likely contributor to the development of Alzheimer's disease. In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in Abeta accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves Abetain vivo, is modified by 4-hydroxy-nonenal (HNE) adducts in the brain of Alzheimer's disease patients. To determine if these changes affected Abeta, SH-SY5Y cells were treated with HNE or Abeta, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted Abeta levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. HNE-modified NEP showed decreased catalytic activity, which was associated with elevations in Abeta1-40 in SH-SY5Y and H4 APP695wt cells. Incubation of cells with Abeta1-42 also induced HNE adduction of NEP. In an apparent compensatory response, Abeta-treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared with the NEP protein level. This study demonstrates that NEP can be inactivated by HNE-adduction, which is associated with, at least partly, reduced Abeta cleavage and enhanced Abeta accumulation.

Cognitive performance correlates with cortical isopeptide immunoreactivity as well as Alzheimer type pathology.

BACKGROUND: Protein cross-linking and aggregation are important molecular processes in Alzheimer's disease (AD), and tissue transglutaminase (tTG) catalyzes protein cross-linking. OBJECTIVES: To measure tTG, tTG enzyme activity and isopeptide, which is the product of tTG, in brain and to relate them to cognitive scores. METHODS: tTG and isopeptide levels were measured in frontal gray matter of 10 normal (NCI), 10 mild cognitive impairment (MCI) and 9 AD brains from the Religious Orders Study. tTG enzymatic activity was measured with a fluorescence assay. RESULTS: tTG protein and enzyme activity were highest in AD, but not significantly greater than MCI or NCI. In contrast, isopeptide immunoreactivity in formic acid extracts was significantly greater in AD than NCI and MCI. The level of insoluble formic acid extractable isopeptide correlated with several measures of cognitive function, including word generation and perceptual speed. Multiple linear regression analyses indicated that insoluble isopeptide immunoreactivity could be accounted for by a combination of factors in the formic acid extract, including Abeta, ubiquitin and tau. CONCLUSIONS: Accumulation of insoluble proteins with isopeptide bonds correlates with cognitive impairment. The relationship of isopeptide to other proteins that are also enriched in formic acid extracts suggests that several substrates of tTG may play a role in the pathogenesis of AD.

The significance of Pin1 in the development of Alzheimer's disease.

Pin1 protein, a peptidyl-prolyl cis-trans isomerase plays an important regulatory role in neuronal function. Recent studies indicate that Pin1 may promote the dephosphorylation of tau and restore its ability to bind to and polymerize microtubles. Previous studies on postmortem human brains showed that Pin1 is down-regulated in advanced Alzheimer's disease (AD) brains compared to age-matched non-demented controls. Because AD is a slowly progressive disease with a preclinical period that can last years, the abundance and regulatory function of Pin1 may vary on the course of the disease. In order to evaluate the potential contribution of Pin1 to AD pathogenesis, levels of mRNA, protein and isomerase activity of Pin1 and phosphorylated tau from postmortem brains of 10 persons with mild-cognitive impairment (MCI), 10 with AD and 10 age-matched no cognitive impairment (NCI) were measured. The relationship between Pin1 and phosphorylated tau as well as clinical and cognitive data were analyzed. The results indicated that Pin1 activity in MCI and AD were significantly higher than in NCI. Phosphorylated tau in MCI and AD was also higher than in NCI group. The positive correlation trend in MCI and the robust correlation in AD between Pin1 activity and phosphorylated tau implies that increasing phosphorylated tau during AD pathogenesis may induce the compensatory activation/up-regulation of Pin1, while the inverse correlation between Pin1 activity and phosphorylated tau in NCI group implies that decreased Pin1 may play a role in the initial accumulation of phosphorylated tau in AD pathogenesis.

Increased frequency of argyrophilic grain disease in Alzheimer disease with 4R tau-specific immunohistochemistry.

Argyrophilic grain disease (AGD) is a medial temporal 4R tauopathy with filamentous inclusions in dendrospinal portions of neurons. AGD is often associated with mild Alzheimer-type pathology, but it is difficult to detect AGD in the setting of advanced Alzheimer disease (AD). The frequency of AGD in AD has been difficult to determine because of masking of grains by neurofibrillary lesions. To address this issue, medial temporal lobe sections from AD brains were immunostained with a 4R tau-specific antibody, ET3, which permitted detection of grains even in the setting of advanced neurofibrillary degeneration. AGD was found in 61 of 239 AD cases (26%). The frequency of AGD in AD in this study is higher than in previous studies that relied on less selective staining methods, such as the Gallyas silver stain or immunostaining with phospho-tau antibodies. The frequency of AGD in AD did not correlate with Braak stage or with the density of neurofibrillary tangles and senile plaques in the limbic lobe; however, AD cases with AGD were significantly older than cases without AGD. The MAPT H1 frequency tended to be higher in AD cases with AGD than in those without AGD, but there were no differences in APOE epsilon4 carrier state. These findings suggest advanced age and possibly MAPT H1 are risk factors for AGD, even in the setting of concurrent AD, in which neurofibrillary degeneration is associated with accumulation of both 3R and 4R tau.

Decreased neprilysin immunoreactivity in Alzheimer disease, but not in pathological aging.

Although evidence suggests that extensive cortical beta-amyloid (Abeta) deposition is essential in Alzheimer disease (AD), it is also detected in nondemented elderly individuals with pathologic aging (PA). Given evidence that neutral endopeptidase (NEP) or neprilysin, a key enzyme for clearance of Abeta, is decreased in AD, the goal of the present study was to determine if NEP was also decreased in PA. We measured NEP immunoreactivity in frontal cortex of 12 AD and six PA cases and compared this with 10 normal (N) elderly individuals. None had any significant other pathology, and they were similar with respect to age, sex, and postmortem delay. In addition, Abeta1-40 and Abeta1-42 were measured by enzyme-linked immunosorbent assay (ELISA), whereas tau, synaptophysin, and alpha-synuclein were measured on Western blots. The AD cases had more neuritic plaques, neurofibrillary tangles, higher Braak stage, and more tau immunoreactivity in frontal cortex than both PA and N. In contrast, both PA and AD had more senile plaques and Abeta1-42 than N. NEP immunoreactivity was decreased in AD but not in PA. The decrease was unlikely the result of neuronal or synaptic loss because NEP immunoreactivity in frontotemporal degeneration with comparable degrees of synaptic loss as the AD cases was not different from control subjects. Although NEP enzyme activity was decreased in approximately half the AD cases, on average, it was not decreased compared with N or PA. The results add further evidence that PA is distinct from AD and indicate that decreased Abeta degradation by NEP is unlikely to contribute significantly to amyloid deposition in PA or, in many cases, of AD.

Neuropathologic, biochemical, and molecular characterization of the frontotemporal dementias.

The frontotemporal dementias (FTDs) are a heterogeneous group of neurodegenerative disorders that are characterized clinically by dementia, personality changes, language impairment, and occasionally extrapyramidal movement disorders. Historically, the diagnosis and classification of FTDs has been fraught with difficulties, especially with regard to establishing a consensus on the neuropathologic diagnosis. Recently, an international group of scientists participated in a consensus conference to develop such neuropathologic criteria. They recommended a diagnostic classification scheme that incorporated a biochemical analysis of the insoluble tau isoform composition, as well as ubiquitin immunohistochemistry. The use and reliability of this classification system has yet to be examined. In this study, we evaluated 21 cases of FTD. Using traditional histochemical stains and tau protein and ubiquitin immunohistochemistry, we separated each case into one of the following categories: classic Pick disease (PiD; n = 7), corticobasal degeneration (CBD; n = 5), dementia lacking distinctive histopathologic features (DLDH; n = 4), progressive supranuclear palsy (PSP; n = 2), frontotemporal lobar degeneration with motor neuron disease or motor neuron disease-type inclusions (FTLD-MND/MNI; n = 2), and neurofibrillary tangle dementia (NFTD; n = 1). Additionally, we independently categorized each case by the insoluble tau isoform pattern, including 3R (n = 5), 4R (n = 7), 3R/4R (n = 3), and no insoluble tau (n = 6). As suggested by the proposed diagnostic scheme, we found that the insoluble tau isoform patterns correlated strongly with the independently derived histopathologic diagnoses (p < 0.001). The data show that cases containing predominantly 3R tau were classic PiD (100%). Cases with predominantly 4R tau were either CBD (71%) or PSP (29%). Cases with both 3R and 4R tau were either a combination of PiD and Alzheimer disease (67%) or NFTD (33%). Finally, cases with no insoluble tau were either DLDH (67%) or FTLD-MND/MNI (33%). To further characterize these cases, we also performed quantitative Western blots for soluble tau, APOE genotyping, and, in selected cases, tau gene sequencing. We show that soluble tau is reduced in DLDH and FTLD-MND/MNI and that APOE4 is overrepresented in PiD and DLDH. We also identified a new family with the R406W mutation and pathology consistent with NFTD. This study validates the recently proposed diagnostic criteria and forms a framework for further refinement of this classification scheme.

From Lewy body disease to Alzheimer's disease: hypothesis and evidence.

The mechanisms underlying Alzheimer's disease and Lewy body disease have long been a controversial subject among neurologists and neuropathologists. Here, we hypothesize that three most common histopathological structures, Lewy body (LB), neurofibrillary tangle (NFT) and neuritic senile plaque (NP) found in neurodegenerative diseases are different stages of the same lesion. Lewy body disease (LBD) and Alzheimer's disease (AD) are based on a similar or the same pathogenic mechanism. Different clinical manifestations and pathological features found in postmortem brains between LBD and AD is due to the different intensities of initial insults, rate of the disease progression and temporal involvement of different brain regions during the AD development.

Contribution of changes in ubiquitin and myelin basic protein to age-related cognitive decline.

The structural substrates for age-associated cognitive and motor slowing are not known, but age-related white matter changes, such as ubiquitin (UBQ)-immunoreactive granular degeneration of myelin, might contribute to this slowing. To address this hypothesis we measured immunoreactivity for UBQ and myelin basic protein (MBP) in frontal white matter of age-, sex- and postmortem interval-matched cases with no cognitive impairment (NCI; N=12), mild cognitive impairment (MCI; N=14) and Alzheimer disease (AD; N=12). There were no significant correlations between UBQ in white matter and cognitive measures, but MBP was significantly lower in AD compared with NCI and MCI. MBP correlated with overall cognition as assessed by neuropsychological summary scores, as well as with timed cognitive tests and those that reflect frontal functions. An age-related decrease in MBP immunoreactivity was detected in NCI cases (r=0.71). These results support the hypothesis that white matter pathology may contribute to age-associated decline in cognition.

Decreases in soluble alpha-synuclein in frontal cortex correlate with cognitive decline in the elderly.

Alpha-synuclein (ASN) is a presynaptic protein and major component of Lewy bodies. It is considered important in the pathogenesis of Alzheimer's disease (AD), but its relevance to progressive cognitive decline in aging is largely unknown. To address this issue, ASN immunoreactivity was measured in frontal cortex of elderly individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and early AD using a Western blot technique and a polyclonal antibody to ASN. ASN immunoreactivity was significantly lower in AD than in MCI and NCI, but there was no difference between MCI and NCI. The ASN immunoreactivity correlated with CERAD diagnosis, as well as Mini-Mental State Exam (MMSE) score, global neuropsychologic z-score and some, but not all, frontal neuropsychology measures. Clinical correlations were stronger for ASN than synaptophysin immunoreactivity assessed in a similar manner. The correlation with MMSE was robust when NCI cases were considered separately, suggesting that decreases in soluble ASN may be an early feature of cognitive decline in aging and AD.

The protection of acetylcholinesterase inhibitor on ß-amyloid-induced the injury of neurite outgrowth via regulating axon guidance related genes expression in neuronal cells.

Cognitive deficits in AD correlate with progressive synaptic dysfunction and loss. The Rho family of small GTPases, including Rho, Rac, and Cdc42, has a central role in cellular motility and cytokinesis. Acetylcholinesterase inhibitor has been found to protect cells against a broad range of reagents-induced injuries. Present studies examined if the effect of HupA on neurite outgrowth in Aß-treated neuronal cells executed via regulating Rho-GTPase mediated axon guidance relative gene expression. Affymetrix cDNA microarray assay followed by real-time RT-PCR and Western Blotting analysis were used to elucidate and analyze the signaling pathway involved in Aß and HupA's effects. The effects of Aß and HupA on the neurite outgrowth were further confirmed via immunofluorescence staining. Aß up-regulated the mRNA expressions of NFAT5, LIMK1, EPHA1, NTN4 and RAC2 markedly in SH-SY5Y cells. Co-incubation of Aß and HupA reversed or decreased the changes of NFAT5, NTN4, RAC2, CDC42 and SEMA4F. HupA treated alone increased NFAT5, LIMK1, NTN4 significantly. Following qRT-PCR validation showed that the correlation of the gene expression ratio between microarray and qRT-PCR is significant. Western blot result showed that the change of CDC42 protein is consistent with the mRNA level while RAC2 is not. The morphological results confirmed that HupA improved, or partly reversed, the Aß-induced damage of neurite outgrowth. The protective effect of HupA from Aß induced morphological injury might be correlative to, at least partially, regulating the network of neurite outgrowth related genes.

Vav1 in hematologic neoplasms, a mini review.

The Vav family of proteins are guanine nucleotide exchange factors which have been shown to be deregulated in several types of human cancer. There are three members of the Vav family that have been identified which are members of the Dbl domain superfamily and have specificity towards Rho/Rac GTPases. The Vav family plays an important role in normal hematologic system development and homeostasis, and Vav1 is largely restricted to the hematologic system. While Vav1 was originally identified as a proto-oncogene, several recent studies have shown that Vav family deletion leads to the development of T-cell malignancies in mice. In addition, Vav1 has been shown to play a role in the ATRA-mediated differentiation of promyelocytic leukemia cells. In this concise review, the gene structure and normal function of Vav1, as well as a possible role for Vav1 in the development of hematologic and other malignancies is reviewed.

TDP-43 in aging and Alzheimer's disease - a review.

Transactive response DNA-binding protein of 43 kDa (TDP-43), an RNA and DNA binding protein involved in transcriptional repression, RNA splicing and RNA metabolism during the stress response, is the major component of neuronal inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions, now referred to as FTLD-TDP. While initially thought to be relatively specific to ALS and FTLD-TDP, TDP-43 pathology has now been detected in a number of other neurodegenerative diseases, many associated with tau pathology, including Guam Parkinson dementia complex and Alzheimer's disease (AD). TDP-43 pathology is detected in 25% to 50% of AD cases, especially those with more severe clinical phenotype and greater Alzheimer type pathology, as well as AD cases with hippocampal sclerosis (HS). HS is characterized by selective neuronal loss affecting CA1 sector of the hippocampus, and most cases of HS, with or without AD, have TDP-43 pathology. Whether TDP-43 pathology is merely an incidental finding in AD or actually contributing to the more severe clinical phenotype remains unresolved. Presence of TDP-43 in normal elderly, who are at increased risk for AD, would strengthen the argument that it is not merely a secondary or incidental finding in end stage AD. Limited studies suggest that TDP-43 pathology is infrequent in neurologically normal elderly (3% or less). We provide an overview of what is known about TDP-43 in AD, normal aging and in other disorders and suggest that TDP-43 proteinopathies be considered in two classes - primary and secondary.

N-acetylcysteine prevents 4-hydroxynonenal- and amyloid-beta-induced modification and inactivation of neprilysin in SH-SY5Y cells.

As one of the dominant amyloid-beta peptide (Abeta) proteases, neprilysin (NEP) plays a crucial role in maintaining a physiologic balance between Abeta production and catabolism. We have previously shown that NEP is modified by 4-hydroxynonenal (HNE) adducts, resulting in decreased activity in the brain of AD patients and cultured cells. In order to determine whether antioxidants can rescue NEP, SH-SY5Y cells were treated with HNE or Abeta, together with N-acetylcysteine for 24 hours, prior to analysis of NEP protein levels, activity, and oxidative modifications. Intracellular NEP developed HNE adducts after 24 hours of HNE or Abeta treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. N-acetylcysteine at 10 to 100 microM alleviated HNE adduction after HNE or Abeta treatment. In keeping with previous reports, HNE-modified NEP showed decreased catalytic activity. The present study demonstrates that antioxidants can be used to spare NEP from oxidative modification, suggesting a potential mechanism underlying the neuroprotective effects of antioxidants in aging or Alzheimer's disease.

Effects of 4-hydroxy-nonenal and Amyloid-beta on expression and activity of endothelin converting enzyme and insulin degrading enzyme in SH-SY5Y cells.

The cerebral accumulation of amyloid-beta (Abeta) is a consistent feature of and likely contributor to the development of Alzheimer's disease (AD). In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism plays an important role in Abeta accumulation. Although endothelin converting enzyme (ECE) and insulin degrading enzyme (IDE) degrade and thus contribute to regulating the steady-state levels of Abeta, how these enzymes are regulated remain poorly understood. In this study, we investigated the effects of 4-hydroxy-nonenal (HNE) and Abeta on the expression and activity of ECE-1 and IDE in human neuroblastoma SH-SY5Y cells. Treatment with HNE or Abeta upregulated ECE-1 mRNA and protein, while IDE was unchanged. Although both ECE-1 and IDE were oxidized within 24 h of HNE or Abeta treatment, ECE-1 catalytic activity was elevated while IDE specific activity was unchanged. The results demonstrated for the first time that both ECE-1 and IDE are substrates of HNE modification induced by Abeta. In addition, the results suggest complex mechanisms underlying the regulation of their enzymatic activity.

Neuroprotective activities of enzymatically hydrolyzed peptides from porcine hide gelatin.

Central nervous system disorders, including cerebrovascular disease, neurodegenerative diseases and head trauma are the most common cause of severe disability in adults and share a number of pathophysiological features. The therapeutic strategy of neuroprotection has been well accepted as one of the promising approaches in treating such brain disorders, and searching for the effective neuroprotective agents is still an open-ended task for neurologists and neuro-pharmacologists. In this study, we report for the first time that the enzymatic hydrolysates from type-B porcine hide gelatin has potent neuroprotective activity against H(2)O(2)- or serum deprivation-induced injuries of cultured SH-SY5Y cells. The peptides used in this study were prepared from type-B porcine hide gelatin digested with pepsin and papain. The neuroprotective activity of the porcine hide gelatin hydrolysate (PHH) was evaluated using MTT reduction assay. From the pre-screening of PHH, we found that the whole porcine hide gelatin hydrolysate obtained from papain digestion (PHH-I) showed significant neuroprotective activities (P<0.05). After further separation of PPH-I through SP-Sephadex C-50 and Sephadex G-25, only the fraction with smaller molecular weight from Sephadex G-25 (PHH-Ic) demonstrated potent neuroprotective activities (P<0.01). The active fraction showed a molecular mass between 1,000-3,000Da in SDS-polyacrylamide gel electrophoresis, and was rich in Glycine, Proline and Hydroxyproline in amino acid composition, indicating that peptides with a spectrum of molecular sizes and certain amino acids are critical for the neuroprotective activities of gelatin peptides. The viability of cultured cells treated with gelatin peptides was significantly improved in a dose-dependent manner. Further studies are necessary to establish the neuroprotective activity of hydrolyzed peptides for the neurons in vivo.

Thymic stromal lymphopoietin (TSLP) as a bridge between infection and atopy.

The rising worldwide prevalence of asthma has intensified interest in the natural history of asthma. An improved understanding of the genetic, environmental, and developmental factors contributing to the inception and exacerbation of asthma will be crucial to efforts to devise effective preventive and therapeutic interventions. There is increasing evidence that the complex interplay of early life respiratory viral infections and allergic sensitization is important in the development of asthma. Major causes of asthma exacerbations are respiratory viral infections and aeroallergen exposure, which may have interactive co-morbid effects. This review describes the potential role of thymic stromal lymphopoietin (TSLP) as a connection between the innate immune response to respiratory viral infections and the type-2 adaptive immune response in the development and exacerbation of asthma.

Tissue transglutaminase, protein cross-linking and Alzheimer's disease: review and views.

Extensive protein cross-linking and aggregation are some of the most common molecular events in the pathogenesis of Alzheimer's disease (AD). Both beta-amyloid (Abeta) plaques and neurofibrillary tangles, which are extracellular and intracellular proteinaceous aggregates, respectively, contribute to neuronal death and progressive cognitive decline. Although protein cross-linking has been recognized and extensively studied for many years, the underlying mechanisms are largely unknown. Recent data indicates that tissue transglutaminase (tTG), which catalyzes the cross-linking of a wide spectrum of proteins including Abeta, tau, alpha-synuclein and neurofilament proteins, may be involved in protein aggregation in AD. Many AD risk factors, such as trauma, inflammation, ischemia and stress, up-regulate tTG protein and activity levels. In this review, we summarize the evidence that tTG plays a role in AD, especially in cross-linking of Abeta, tau, alpha-synuclein and neurofilament proteins. An experimentally testable hypothesis is that tTG may play a central role in AD pathogenesis and that it provides a conceptual link between sporadic and familial AD through a shared pathogenic pathway.

beta-Amyloid degradation and Alzheimer's disease.

Extensive beta-amyloid (A beta) deposits in brain parenchyma in the form of senile plaques and in blood vessels in the form of amyloid angiopathy are pathological hallmarks of Alzheimer's disease (AD). The mechanisms underlying A beta deposition remain unclear. Major efforts have focused on A beta production, but there is little to suggest that increased production of A beta plays a role in A beta deposition, except for rare familial forms of AD. Thus, other mechanisms must be involved in the accumulation of A beta in AD. Recent data shows that impaired clearance may play an important role in A beta accumulation in the pathogenesis of AD. This review focuses on our current knowledge of A beta-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), insulin-degrading enzyme (IDE), angiotensin-converting enzyme (ACE), and the plasmin/uPA/tPA system as they relate to amyloid deposition in AD.

Molecular cloning and characterization of rat and human calpain-5.

Until today, 14 isoforms of mammalian calpains have been identified, including calpain-5. The C. elegans calpain-5 homologue tra-3 is reported to be essential for necrotic neuronal cell death. In this study, we cloned and characterized rat calpain-5, which is highly homologous to human and mouse sequences. The nucleotide sequence is 87% and 93% identical with human and mouse calpain-5, respectively. The protein sequence is well conserved, showing 96% identity in mouse and 92% in human. RT-PCR analysis revealed strong expression of calpain-5 in rat lungs, kidneys, and brain while week expression in heart, whereas in rat brain regions it is ubiquitously expressed. The mRNA expression in different human tissues showed equal expression. However, in human brain regions calpain-5 was strongly expressed in hypothalamus, thalamus, cerebellum, and frontal lobe. Western blot analysis on human neuroblastoma SH-SY5Y cells demonstrated calcium-dependent processing of calpain-5, despite the absence of calmodulin-like domain.