Targeting Alzheimer's disease with gene and cell therapies.

Alzheimer's disease (AD) causes dementia in both young and old people affecting more than 40 million people worldwide. The two neuropathological hallmarks of the disease, amyloid beta (Aß) plaques and neurofibrillary tangles consisting of protein tau are considered the major contributors to the disease. However, a more complete picture reveals significant neurodegeneration and decreased cell survival, neuroinflammation, changes in protein and energy homeostasis and alterations in lipid and cholesterol metabolism. In addition, gene and cell therapies for severe neurodegenerative disorders have recently improved technically in terms of safety and efficiency and have translated to the clinic showing encouraging results. Here, we review broadly current data within the field for potential targets that could modify AD through gene and cell therapy strategies. We envision that not only Aß will be targeted in a disease-modifying treatment strategy but rather that a combination of treatments, possibly at different intervention times may prove beneficial in curing this devastating disease. These include decreased tau pathology, neuronal growth factors to support neurons and modulation of neuroinflammation for an appropriate immune response. Furthermore, cell based therapies may represent potential strategies in the future.

Soft-diet feeding after weaning affects behavior in mice: Potential increase in vulnerability to mental disorders.

Mastication is one of the most important oral functions, and the period during which mastication is acquired overlaps with the term of rapid development and maturation of the neural systems. In particular, the acquisition period after weaning is related to the potential onset of mental disorders. However, the roles of mastication during this period for brain development remain largely unknown. Therefore, we used a series of standard behavioral analyses, assessment of hippocampal cell proliferation, and the expression of brain-derived neurotrophic factor (BDNF), TrkB, and Akt1 in the hippocampus and frontal cortex of mice to investigate the effects of post-weaning mastication on brain function. We fed 21-day-old C57BL6/J male mice either a hard or a soft diet for 4weeks and conducted a series of standard behavioral tests from 7weeks of age. Further, histological analysis with bromodeoxyuridine was performed to compare hippocampal cell proliferation at 7 and 14weeks of age. Real-time polymerase chain reaction was performed to compare BDNF, TrkB, and Akt1 expression in the hippocampus and frontal cortex of 14-week-old mice. Compared to mice fed a hard diet (HDM), soft-diet mice (SDM) showed behavioral impairments, including decreased home cage activity, increased open field test activity, and deficits in prepulse inhibition. These results were similar to those observed in mouse models of schizophrenia. However, no effects were observed on anxiety-like behaviors or memory/learning tests. Compared to HDM, SDM showed significantly decreased hippocampal cell proliferation and hippocampal BDNF and Akt1 gene expression at 14weeks of age. A soft diet after weaning may have resulted in histological and molecular changes in the hippocampus and influenced outcomes of behavioral tests related to mental disorders. Our findings suggest that soft-diet feeding after weaning may affect both physical and mental development of mice, and may increase vulnerability to mental disorders.

Etiology of sporadic Alzheimer's disease: somatostatin, neprilysin, and amyloid beta peptide.

We recently demonstrated that amyloid beta peptide (Abeta) is catabolized primarily by a neutral endopeptidase, neprilysin, in the brain and that a neuropeptide, somatostatin (SST), regulates brain Abeta level via modulation of neprilysin activity. Because SST expression in the brain declines upon aging in various mammals including rodents, apes and humans, we hypothesize that the aging-dependent reduction of SST triggers accumulation of Abeta in the brain by suppressing neprilysin action. This hypothesis accounts for the fact that aging is the predominant risk factor for Sporadic Alzheimer's disease.

Novel amyloid precursor protein gene missense mutation (D678N) in probable familial Alzheimer's disease.

OBJECTIVE: To describe a novel missense mutation, Asp678Asn (D678N), in the amyloid precursor protein (APP) gene in a Japanese pedigree of probable familial Alzheimer's disease (FAD). SUBJECT: The proband was a women of 72. Symptoms of dementia that fulfilled the criteria for probable Alzheimer's disease appeared at about 60 years of age, and slowly worsened over more than 10 years without evident cerebrovascular complications, either clinically or neuroradiologically. METHODS: Polymerase chain reaction single strand conformational polymorphism (PCR-SSCP) analysis followed by sequence analysis was used to examine genomic DNA of the proband for mutations in the APP gene exons 16 and 17. RESULTS: Analysis of the APP exon 16 in the proband showed a GAC to AAC nucleotide substitution in codon 678 of the APP gene, causing an amino acid substitution of Asp to Asn (D678N). Heterozygosity of the APP D678N mutation was found in the proband and in the demented elder sister. CONCLUSIONS: The production and accumulation of mutated Abeta (Asn7-Abeta) or the misfunction of D678N mutant APP may have pathogenic properties for the development of Alzheimer's disease in this pedigree.

beta2-Adrenergic receptor responsiveness of the calpain-calpastatin system and attenuation of neuronal death in rat hippocampus after transient global ischemia.

In the CNS, where Ca(2+) overload has been established as a mechanism contributing to neuronal damage associated with excitotoxicity, stroke and ischemia, there is interest in understanding the role of calpain inhibition in rescuing neurons from death. In these settings, the activation of large stores of latent calpain may rapidly lead to the demise of the neuron within hours. The activity of calpain is strictly regulated by calcium concentrations and interactions with calpastatin (endogenous calpain inhibitor). The interaction between calpains and calpastatin is calcium dependent, and little is known about the regulation of the neuronal calpain-calpastatin system in vivo. It has been postulated that calpastatin can be modulated by nerve growth factors (NGFs). We have demonstrated in vitro as well as in vivo a neuroprotective effect of the beta(2)-adrenoceptor agonist clenbuterol (CLN) mediated through an increased NGF expression. In this study we attempt to find out whether CLN is capable (1) of modulating proteolysis regulated by the calpain-calpastatin system and (2) of attenuating DNA-fragmentation induced by cerebral ischemia. Rats received CLN daily for 1 week, were then subjected to ischemia and finally perfused at different times post-ischemia. The proteolytic activity of calpain was measured by the immunolocalisation of calpastatin and spectrin-breakdown products (SBP). The time course of apoptosis was assessed by terminal dUTP nick end-labeling (TUNEL)-staining. CLN reduced CA1-hippocampal cell damage by 23%, attenuated DNA-laddering and decreased proteolysis of spectrin by enhancing calpastatin activity. These results provide evidence that CLN is a potent neuroprotective substance, which through the enhancement of calpastatin synthesis attenuates the apoptotic machinery and modulates proteolysis.

Calpain-dependent proteolysis of merlin occurs by oxidative stress in meningiomas: a novel hypothesis of tumorigenesis.

BACKGROUND: The purpose of this study is to indicate that oxidative stress may contribute to occurrence of meningiomas. Recently, it was reported that aside from the neurofibromatosis type 2 (NF2) gene mutations, the calpain-dependent proteolysis of the NF2 gene product, merlin might be closely related to the development of certain NF2-related tumors. Although meningiomas are well known to occur more frequently in aged persons, it still remains unknown why calpain activation occurs predominantly in them. Because the production of free radicals with aging might be one of the causes of calpain activation especially in leptomeningeal cells being devoid of blood supply, the authors examined the relations between mu-calpain activation and merlin proteolysis induced by the oxidative stress. METHODS: The authors examined 12 patient-derived sporadic meningiomas and their primary cultured cells. Malignant glioma cell line (U-251MG), which had no relation to NF2, was used as a control. They were exposed to hydrogen peroxide (H2O2) for 1 hour. After oxidative stress, they were examined by Western blot and immunofluorescence microscopic analyses. RESULTS: Despite the consistent expressions of activated mu-calpain in 11 of 12 meningioma tissues, this calpain activation completely disappeared after culture; instead the full-length merlin appeared again in 8 of 11 cases. The treatment of cultured cells with hydrogen peroxide induced both mu-calpain-dependent cleavage of merlin and reduction of an intrinsic calpain inhibitor calpastatin. Such proteolysis was significantly blocked by a specific calpain inhibitor, Z-LLal. The full-length merlin was immunocytochemically colocalized with activated mu-calpain at the plasma membrane, and, after mu-calpain activation, the fragment of merlin translocated to the perinuclear cytoplasm or into the nucleus. CONCLUSIONS: These findings suggest that oxidative stress-induced activation of mu-calpain causes proteolysis of merlin conceivably to impair cell adhesion and/or contact inhibition of meningioma cells.

Clearance of extracellular and cell-associated amyloid beta peptide through viral expression of neprilysin in primary neurons.

Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain. We previously demonstrated that neprilysin is the major Abeta-degrading enzyme in vivo. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating Abeta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on Abeta metabolism. The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wild-type neprilysin, but not the mutant, led to significant decreases in both the Abeta40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expression also resulted in reducing cell-associated Abeta, which could be more neurotoxic than extracellular Abeta. These results indicate that the manipulation of neprilysin activity in neurons, the major source of Abeta in the brain, would be a relevant strategy for controlling the Abeta levels and thus the Abeta-associated pathology in brain tissues.

Temporospatial sequence of cellular events associated with etoposide-induced neuronal cell death: role of antiapoptotic protein Bcl-X(L).

Etoposide-induced death comprises such nuclear events as the formation of topoisomerase II-DNA cleavable complex and cytosolic events including caspase activation. By first establishing the temporospatial death sequence triggered by etoposide in a neuronal cell line, MN9D overexpressing Bcl-X(L) (MN9D/Bcl-X(L)) or control vector (MN9D/Neo), we examined whether formation of this complex is primarily responsible for cell death and at which strategic points and how Bcl-X(L) blocks etoposide-induced neuronal death. Etoposide induced death that was dependent on caspase, cycloheximide, and calpain in MN9D/Neo cells. Etoposide also induced death in enucleated MN9D/Neo cells, although this was less severe. The level of topoisomerase II-DNA cleavable complex reached at a maximum of 2 hr after etoposide treatment was identical in MN9D/Neo and MN9D/Bcl-X(L) cells. In MN9D/Neo cells, cytochrome c release into the cytosol and caspase activation occurred as early as 2 hr and 3-6 hr after etoposide treatment, respectively. Etoposide-induced DNA laddering potentially via caspase appeared as early as 12 hr after drug treatment, followed by nuclear swelling in MN9D/Neo cells (>18-20 hr). Subsequently, nuclear condensation started by 24-28 hr and became apparent thereafter. All of these events except for nuclear swelling were substantially blocked in MN9D/Bcl-X(L). At the later stage of cell death (<32-36 hr), a specific cleavage of Bax and fodrin appeared that was completely blocked by calpain inhibitor or by Bcl-X(L). Taken together, our data suggest that Bcl-X(L) prevents etoposide-induced neuronal death by exerting its anticaspase and anticalpain effect on cellular events after the formation of topoisomerase II-DNA cleavable complex that may not be a major contributor to cell death.

Alzheimer's beta-secretase, beta-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase.

The deposition of amyloid beta-peptide (A beta) in the brain is closely associated with the development of Alzheimer's disease. A beta is generated from the amyloid precursor protein (APP) by sequential action of beta-secretase (BACE1) and gamma-secretase. Although BACE1 is distributed among various other tissues, its physiological substrates other than APP have yet to be identified. ST6Gal I is a sialyltransferase that produces a sialyl alpha 2,6galactose residue, and the enzyme is secreted out of the cell after proteolytic cleavage. We report here that BACE1 is involved in the proteolytic cleavage of ST6Gal I, on the basis of the following observations. ST6Gal I was colocalized with BACE1 in the Golgi apparatus by immunofluorescence microscopy, suggesting that BACE1 acts on ST6Gal I within the same intracellular compartment. When BACE1 was overexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I markedly increased. When APP(SW) (Swedish familial Alzheimer's disease mutation), a preferable substrate for BACE1, was coexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I significantly decreased, suggesting that that the beta-cleavage of overexpressed APP(SW) competes with ST6Gal I processing. In addition, BACE1-Fc (Fc, the hinge and constant region of IgG) chimera cleaved protein A-ST6Gal I fusion protein in vitro. Thus, we conclude that BACE1 is responsible for the cleavage and secretion of ST6Gal I.

Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2.

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress-specific protease activation pathway exists, and to what extent Bcl-2 plays a role in preventing drug-induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase-dependent apoptosis while a dopaminergic neurotoxin, MPP(+) largely induced caspase-independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS- and MPP(+)-induced cell death, Bax was cleaved into an 18-kDa fragment. This 18-kDa fragment appeared only in the mitochondria-enriched heavy membrane fraction of STS-treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP(+)-treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [(35)S]methionine-labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP(+)- and STS-induced Bax cleavage. Intriguingly, overexpression of baculovirus-derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS- but not MPP(+)-induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP(+)-induced but not from STS-induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl-2 prevented both STS- and MPP(+)-induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl-2 may play a protective role by primarily blocking drug-induced caspase or calpain activity in dopaminergic neuronal cells.

Metabolic regulation of brain Abeta by neprilysin.

Amyloid beta peptide (Abeta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite in the brain. We examined the role of neprilysin, a candidate Abeta-degrading peptidase, in the metabolism using neprilysin gene-disrupted mice. Neprilysin deficiency resulted in defects both in the degradation of exogenously administered Abeta and in the metabolic suppression of the endogenous Abeta levels in a gene dose-dependent manner. The regional levels of Abeta in the neprilysin-deficient mouse brain were in the distinct order of hippocampus, cortex, thalamus/striatum, and cerebellum, where hippocampus has the highest level and cerebellum the lowest, correlating with the vulnerability to Abeta deposition in brains of humans with AD. Our observations suggest that even partial down-regulation of neprilysin activity, which could be caused by aging, can contribute to AD development by promoting Abeta accumulation.

Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains.

Tau in Alzheimer neurofibrillary tangles has been shown to be hyperphosphorylated and CDK5, GSK3, MAP kinase and SAP kinases are the candidate kinases for the phosphorylation of tau. Recently, it was reported that the conversion of p35, the activator of CDK5, to p25 was upregulated in Alzheimer's disease (AD) brains, and that p35 is cleaved to yield p25 by calpain. Here we show that p35 is rapidly cleaved to p25 in rat and human brains within a short postmortem delay and that the conversion of p35 to p25 is partially dependent on calpain activity. Immunoblot analysis of brains prepared from patients with AD or age-matched control individuals with a short postmortem delay revealed no specific increase in the levels of p25 in AD brains, whereas the levels of active form of calpain were increased in AD brains compared to the those in controls. These observations suggest that the conversion of p35 to p25 is a postmortem degradation event and may not be upregulated in AD brains.

Matrix metalloproteinase (MMP) system in brain: identification and characterization of brain-specific MMP highly expressed in cerebellum.

The matrix metalloproteinase (MMP) family, comprising more than 20 isoforms, modulates the extracellular milieu by degrading extracellular matrix (ECM) proteins. Because MMP is one of the few groups of proteinases capable of hydrolysing insoluble fibrillar proteins, they are likely to play crucial roles in regulating both normal and pathophysiological processes in the brain. However, little is yet known about their possible neuronal functions due presumably to their unusual redundancy and to the absence of a complete catalogue of isoforms. As an initial step in understanding the MMP system in the brain, we analysed an expression spectrum of MMP in rat brain using RT-PCR and discovered a novel brain-specific MMP, MT5-MMP. MT5-MMP was the predominant species among the nongelatinase-type isoforms in brain. MT5-MMP, present in all brain tissues examined, was most strongly expressed in cerebellum and was localized in the membranous structures of expressing neurons, as assessed biochemically and immunohistochemically. In cerebellum, its expression was regulated developmentally and was closely associated with dendritic tree formation of Purkinje cells, suggesting that MT5-MMP may contribute to neuronal development. Furthermore, its stable postdevelopmental expression and colocalization with senile plaques in Alzheimer brain indicates possible roles in neuronal remodeling naturally occurring in adulthood and in regulating pathophysiological processes associated with advanced age.

Neprilysin degrades both amyloid beta peptides 1-40 and 1-42 most rapidly and efficiently among thiorphan- and phosphoramidon-sensitive endopeptidases.

To identify the amyloid beta peptide (Abeta) 1-42-degrading enzyme whose activity is inhibited by thiorphan and phosphoramidon in vivo, we searched for neprilysin (NEP) homologues and cloned neprilysin-like peptidase (NEPLP) alpha, NEPLP beta, and NEPLP gamma cDNAs. We expressed NEP, phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PEX), NEPLPs, and damage-induced neuronal endopeptidase (DINE) in 293 cells as 95- to 125-kDa proteins and found that the enzymatic activities of PEX, NEPLP alpha, and NEPLP beta, as well as those of NEP and DINE, were sensitive to thiorphan and phosphoramidon. Among the peptidases tested, NEP degraded both synthetic and cell-secreted Abeta1-40 and Abeta1-42 most rapidly and efficiently. PEX degraded cold Abeta1-40 and NEPLP alpha degraded both cold Abeta1-40 and Abeta1-42, although the rates and the extents of the digestion were slower and less efficient than those exhibited by NEP. These data suggest that, among the endopeptidases whose activities are sensitive to thiorphan and phosphoramidon, NEP is the most potent Abeta-degrading enzyme in vivo. Therefore, manipulating the activity of NEP would be a useful approach in regulating Abeta levels in the brain.

Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease.

The accumulation of amyloid beta protein (Abeta) in the Tg2576 mouse model of Alzheimer's disease (AD) was evaluated by ELISA, immunoblotting, and immunocytochemistry. Changes in Abeta begin at 6-7 months as SDS-insoluble forms of Abeta42 and Abeta40 that require formic acid for solubilization appear. From 6 to 10 months, these insoluble forms increase exponentially. As insoluble Abeta appears, SDS-soluble Abeta decreases slightly, suggesting that it may be converting to an insoluble form. Our data indicate that it is full-length unmodified Abeta that accumulates initially in Tg2576 brain. SDS-resistant Abeta oligomers and most Abeta species that are N-terminally truncated or modified develop only in older Tg2576 mice, in which they are present at levels far lower than in human AD brain. Between 6 and 10 months, when SDS-insoluble Abeta42 and Abeta40 are easily detected in every animal, histopathology is minimal because only isolated Abeta cores can be identified. By 12 months, diffuse plaques are evident. From 12 to 23 months, diffuse plaques, neuritic plaques with amyloid cores, and biochemically extracted Abeta42 and Abeta40 increase to levels like those observed in AD brains. Coincident with the marked deposition of Abeta in brain, there is a decrease in CSF Abeta and a substantial, highly significant decrease in plasma Abeta. If a similar decline occurs in human plasma, it is possible that measurement of plasma Abeta may be useful as a premorbid biomarker for AD.

Biochemical identification of the neutral endopeptidase family member responsible for the catabolism of amyloid beta peptide in the brain.

Amyloid beta peptide (Abeta) is a physiological peptide that is constantly catabolized in the brain. We previously demonstrated that an endopeptidase sensitive to phosphoramidon and thiorphan conducts the initial rate-limiting proteolysis of Abeta in vivo, but the exact molecular identity of the peptidase(s) has remained unknown because of the molecular redundancy of such activity. We analyzed the brain-derived enzyme by means of immuno-depletion and gene disruption, and demonstrate here that neprilysin accounts for the majority of the Abeta-degrading activity. Furthermore, kinetic analysis, giving a K(m) value of 2.8 +/- 0.76 microM, indicated that Abeta(1-42) is a relevant substrate for neprilysin.

Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active.

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved beta3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s).

Amyloid beta protein starting pyroglutamate at position 3 is a major component of the amyloid deposits in the Alzheimer's disease brain.

The amyloid beta protein (Abeta) deposited in the Alzheimer's disease (AD) brain is heterogeneous at both its amino and carboxyl termini. Recent studies of the genetic forms of AD indicate that the aggregation and deposition of Abeta42 may be a common initiating event in all forms of AD. Here, we analyzed the amino termini of the Abeta species deposited in the AD brain, focusing specifically on species with amino-terminal pyroglutamate at position 3 (Abeta3(pE)). Immunocytochemical analysis of AD brains with an antibody specific for Abeta3(pE) confirmed that these species deposit in blood vessels and senile plaques. Using specific sandwich ELISAs, we determined the amounts of Abeta3(pE)-40 and Abeta3(pE)-42(43) in AD brain compared with other forms. This analysis showed that Abeta3(pE)-40 is closely correlated with the extent of Abeta deposition in blood vessels, whereas Abeta3(pE)-42(43) is not. In addition, Abeta3(pE)-42(43) is an important component of the Abeta deposited in senile plaques of the AD brain, constituting approximately 25% of the total Abeta42(43). In vitro comparison of Abeta1-42 and Abeta3(pE)-42 showed that Abeta3(pE)-42 is highly prone to oligomerization. These findings suggest that Abeta3(pE)-42 may be particularly important in AD pathogenesis.

Molecular cloning and expression of aminopeptidase A isoforms from rat hippocampus.

The full-length cDNA encoding aminopeptidase A (APAL) was cloned from a rat hippocampus cDNA library. A short variant aminopeptidase A (APAS), produced by deletion, was also cloned. In the case of APAL, the longest open reading frame encodes 945 amino acid residues with a calculated molecular mass of 108 kDa, and the deduced amino acid sequence shows 76, 86 and 78% identity with its human, murine and porcine counterparts, respectively. Rat aminopeptidase A mRNAs were detected in the kidney, liver, heart and brain by Northern blot analysis. When overexpressed in COS-1 cells, APAL shows apparent aminopeptidase A activity, whereas APAS does not.