A conserved MTMR lipid phosphatase increasingly suppresses autophagy in brain neurons during aging.

Ageing is driven by the progressive, lifelong accumulation of cellular damage. Autophagy (cellular self-eating) functions as a major cell clearance mechanism to degrade such damages, and its capacity declines with age. Despite its physiological and medical significance, it remains largely unknown why autophagy becomes incapable of effectively eliminating harmful cellular materials in many cells at advanced ages. Here we show that age-associated defects in autophagic degradation occur at both the early and late stages of the process. Furthermore, in the fruit fly Drosophila melanogaster, the myotubularin-related (MTMR) lipid phosphatase egg-derived tyrosine phosphatase (EDTP) known as an autophagy repressor gradually accumulates in brain neurons during the adult lifespan. The age-related increase in EDTP activity is associated with a growing DNA N6-adenine methylation at EDTP locus. MTMR14, the human counterpart of EDTP, also tends to accumulate with age in brain neurons. Thus, EDTP, and presumably MTMR14, promotes brain ageing by increasingly suppressing autophagy throughout adulthood. We propose that EDTP and MTMR14 phosphatases operate as endogenous pro-ageing factors setting the rate at which neurons age largely independently of environmental factors, and that autophagy is influenced by DNA N6-methyladenine levels in insects.

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

AUTEN-67 (Autophagy Enhancer-67) Hampers the Progression of Neurodegenerative Symptoms in a Drosophila model of Huntington's Disease.

BACKGROUND: Autophagy, a lysosome-mediated self-degradation process of eukaryotic cells, serves as a main route for the elimination of cellular damage [1-3]. Such damages include aggregated, oxidized or misfolded proteins whose accumulation can cause various neurodegenerative pathologies, including Huntington's disease (HD). OBJECTIVE: Here we examined whether enhanced autophagic activity can alleviate neurophatological features in a Drosophila model of HD (the transgenic animals express a human mutant Huntingtin protein with a long polyglutamine repeat, 128Q). METHODS: We have recently identified an autophagy-enhancing small molecule, AUTEN-67 (autophagy enhancer 67), with potent neuroprotective effects [4]. AUTEN-67 was applied to induce autophagic activity in the HD model used in this study. RESULTS: We showed that AUTEN-67 treatment interferes with the progressive accumulation of ubiquitinated proteins in the brain of Drosophila transgenic for the pathological 128Q form of human Huntingtin protein. The compound significantly improved the climbing ability and moderately extended the mean life span of these flies. Furthermore, brain tissue samples from human patients diagnosed for HD displayed increased levels of the autophagy substrate SQSTM1/p62 protein, as compared with controls. CONCLUSIONS: These results imply that AUTEN-67 impedes the progression of neurodegenerative symptoms characterizing HD, and that autophagy is a promising therapeutic target for treating this pathology. In humans, AUTEN-67 may have the potential to delay the onset and decrease the severity of HD.

Capsaicin promotes the amyloidogenic route of brain amyloid precursor protein processing.

Besides being an important component of spices used worldwide, capsaicin has wide-ranging therapeutic potential as a hypolipidemic, antioxidant and anti-inflammatory agent. Accordingly, it is very important to investigate the long-term effect of capsaicin in the pathogenesis of Alzheimer's disease. In this study, the effects of capsaicin on the processing of amyloid precursor protein (APP) were investigated in an in vivo model. The APP mRNA and protein levels were examined in the brain cortices of control and capsaicin-treated rats. The protein kinase C (PKC) translocation state in the soluble and membrane-bound fractions and the levels of beta-secretase (BACE) were also evaluated. Capsaicin enhanced the level of membrane-bound APP 1.7-fold. The APP mRNA and PKC and BACE protein levels were unchanged after capsaicin treatment. These in vivo data indicate that capsaicin is able to interfere with the brain APP metabolism by promoting the amyloidogenic route. We suggest that PKC is not involved in the mechanism underlying the effects.

APP mRNA splicing is upregulated in the brain of biglycan transgenic mice.

Many of the risk factors for cerebrovascular disease and atherosclerosis also increase the risk of Alzheimer's disease, characterized by the cerebral deposition of beta-amyloid plaques resulting from the abnormal processing of the transmembrane amyloid precursor protein (APP). The initiating event of cholesterol-induced atherosclerosis is the retention and accumulation of atherogenic apolipoprotein B (apoB) together with low-density lipoproteins in the vascular intima. Biglycan, a member of the small leucine-rich protein family, was suspected of contributing to this process. The individual and combined overexpressions of biglycan and apoB-100 were therefore examined on the cortical APP mRNA levels of transgenic mice by means of semiquantitative PCR. As compared with the control littermates, transgenic biglycan mice had significantly increased cortical APP695 (122%) and APP770 (157%) mRNA levels, while the double transgenic (apoB(+/-)xbiglycan(+/-)) mice did not exhibit any changes. These results provide the first experimental evidence that the atherogenic risk factor biglycan alters APP splicing and may participate in the pathogenesis of both Alzheimer and vascular dementias.

3,4-Methylenedioxymethamphetamine (MDMA), but not morphine, alters APP processing in the rat brain.

The abuse of drugs such as opioids and 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') can have detrimental effects on the cognitive functions, but the exact molecular mechanism whereby these drugs promote neurodegeneration remains to be elucidated. The major purpose of the present pilot study was to determine whether the chronic in-vivo administration of morphine (10 mg/kg) or MDMA (1 mg/kg) to rats can alter the expression and processing of amyloid precursor protein (APP), the central molecule in the proposed pathomechanism of Alzheimer's disease. MDMA treatment significantly decreased the production of APP in the cytosolic fraction of the brain cortex. A concomitant 25% increase was found both in the beta-secretase (BACE) and APP mRNA levels (108%). In contrast, in the applied single dosage chronic morphine treatment did not influence either the APP and BACE protein levels or the APP mRNA production. These results indicate that the chronic use of 'ecstasy', but not morphine, may be harmful via a novel mode of action, i.e. by altering the APP expression and processing in the brain.

Age-dependent oxidative stress-induced DNA damage in Down's lymphocytes.

The aim of the present study was to investigate the oxidative status of lymphocytes from children (n=7) and adults (n=18) with Down's syndrome (DS). The basal oxidative condition, the vulnerability to in vitro hydrogen peroxide exposure, and the repair capacity were measured by means of the damage-specific alkaline comet assay. Significantly and age-independently elevated numbers of single strand breaks and oxidized bases (pyrimidines and purines) were found in the nuclear DNA of the lymphocytes in the DS group in the basal condition. These results may support the role of an increased level of endogenous oxidative stress in DS and are similar to those previously demonstrated in Alzheimer's disease. In the in vitro oxidative stress-induced state, a markedly higher extent of DNA damage was observed in DS children as compared with age- and gender-matched healthy controls, suggesting that young trisomic lymphocytes are more sensitive to oxidative stress than normal ones. However, the repair ability itself was not found to be deteriorated in either DS children or DS adults.

Human apoB overexpression and a high-cholesterol diet differently modify the brain APP metabolism in the transgenic mouse model of atherosclerosis.

Epidemiological and biochemical data suggest a link between the cholesterol metabolism, the amyloid precursor protein (APP) processing and the increased cerebral beta-amyloid (Abeta) deposition in Alzheimer's disease (AD). The individual and combined effects of a high-cholesterol (HC) diet and the overexpression of the human apoB-100 gene were therefore examined on the cerebral expression and processing of APP in homozygous apoB-100 transgenic mice [Tg (apoB(+/+))], a validated model of atherosclerosis. When fed with 2% cholesterol for 17 weeks, only the wild-type mice exhibited significantly increased APP695 (123%) and APP770 (138%) mRNA levels in the cortex. The HC diet-induced hypercholesterolemia significantly increased the APP isoform levels in the membrane-bound fraction, not only in the wild-type animals (114%), but also in the Tg apoB(+/+) group (171%). The overexpression of human apoB-100 gene by the liver alone reduced the brain APP isoform levels in the membrane-bound fraction (78%), whereas the levels were increased by the combined effect of HC and the overexpression of the human apoB-100 gene (134%). The protein kinase C and beta-secretase protein levels were not altered by the individual or combined effects of these two factors. Our data indicate that the two atherogenic factors, the HC diet and the overexpression of the human apoB-100 gene by the liver, could exert different effects on the processing and expression of APP in the mice brain.

Alzheimer's lymphocytes are resistant to ultraviolet B-induced apoptosis.

In the present pilot investigation, the susceptibility of T-lymphocytes from Alzheimer's disease (AD) subjects (n=22) and aged-matched, non-demented controls (CNT) (n=12) was examined with ultraviolet (UV) B light-induced apoptosis in vitro. The basal apoptotic ratios were similar in both groups. However, the AD lymphocytes displayed significantly (p<0.0001) lower apoptotic levels than those of the CNT lymphocytes at all of the applied UVB exposure doses (100, 200 and 300 mJ/cm(2)). These observations indicate that AD lymphocytes are more resistant than CNT lymphocytes to UVB irradiation.

CYP46 T/C polymorphism is not associated with Alzheimer's dementia in a population from Hungary.

Multiple genetic and environmental factors regulate the susceptibility to Alzheimer's disease (AD). Recently, several independent studies have reported that a locus on chromosome 14q32.1, where a gene encoding a cholesterol degrading enzyme of the brain, called 24-hydroxylase (CYP46A1) is located, has been linked with AD. The single nucleotide polymorphism (T/C) in intron 2 of CYP46 gene has been found to confer the risk for AD. The water soluble 24(S)-hydroxysterol is the product of the CYP46A1, and elevated plasma and cerebrospinal fluid hydroxysterol concentrations have been found in AD, reflecting increased brain cholesterol turnover or cellular degradation, due to the neurodegenerative process. A case-control study was performed on 125 AD and 102 age- and gender-matched control subjects (CNT) from Hungary, to test the association of CYP46 T/C and apolipoprotein E (ApoE) gene polymorphisms in AD. The frequency of the CYP46 C allele was similar (chi2=0.647, df=1, P=0.421, exact P=0.466, OR=0.845; 95% CI: 0.561-1.274) in both groups (CNT: 27%; 95% CI: 21.3-33.4; AD 30%; 95% CI: 25.0-36.3). The ApoE varepsilon4 allele was significantly over-represented (chi2=11.029, df=2, P=0.004) in the AD population (23.2%; 95% CI: 18.2-29.0) when compared with the CNT (11.3%; 95% CI: 7.4-16.6). The presence or absence of one or two CYP46C alleles together with the ApoE varepsilon4 allele did not increase the risk of AD (OR=3.492; 95% CI: 1.401-8.707; P<0.007 and OR=3.714; 95% CI: 1.549-8.908; P<0.003, respectively). Our results indicate that the intron 2 T/C polymorphism of CYP46 gene (neither alone, nor together with the varepsilon4 allele) does not increase the susceptibility to late-onset sporadic AD in the Hungarian population.

Effect of general anesthetics on amyloid precursor protein and mRNA levels in the rat brain.

The incidence of Alzheimer's disease is elevated after exposure to surgical interventions. Since amyloid precursor protein (APP) and its neurotoxic derivatives play key roles in the development of Alzheimer dementia, the role of general anesthesia is controversial in the development of cognitive decline. As such, the effect of anesthetics on APP protein and mRNA levels was assessed utilizing semiquantitative Western-immunoblot and reverse transcription polymerase chain reaction (RT-PCR) in brains of rats following intraperitoneal treatment with propofol and thiopental. The anesthetics did not change cortical APP protein and mRNA concentration considerably. These results indicate that both propofol and thiopental are considered to be relatively safe with respect to APP metabolism.

Imipramine and citalopram facilitate amyloid precursor protein secretion in vitro.

Comorbid depression of Alzheimer's disease (AD) is a common mood disorder in the elderly and a broad spectrum of antidepressants have been used for its treatment. Abeta peptides and other derivatives of the amyloid precursor protein (APP) have been implicated as central to the pathogenesis of AD. However, the functional relationship of APP and its proteolytic derivatives to antidepressant therapy is not known. In this study, Western blotting was used to test the ability of the tricyclic antidepressant (TCA) imipramine or the selective serotonin reuptake inhibitor (SSRI) citalopram to change the release of APP and the protein kinase C (PKC) content. Both antidepressants increased APP secretion in primary rat neuronal cultures. Imipramine or citalopram enhanced the level of secreted APP by 3.2- or 3.4-fold, respectively. Increases in PKC level were observed only after imipramine treatment. These in vitro data suggest that both TCA and SSRI are able to interfere with the APP metabolism. Imipramine promotes the non-amyloidogenic route of APP processing via stimulatory effects on PKC. We propose that PKC is not involved in the mechanism underlying the effects of citalopram on the APP metabolism. Since the secreted APP is not further available for the pathological cleavage of beta- and gamma-secretases, antidepressant medication might be beneficial in AD therapy.

Impact of haloperidol and risperidone on gene expression profile in the rat cortex.

Despite the clinical efficacy of the most thoroughly studied conventional neuroleptic agent haloperidol, and the atypical antipsychotic risperidone is well established, little information is available on their molecular effects. Recent advances in high-density DNA microarray techniques allow the possibility to analyze thousands of genes simultaneously for their differential gene expression patterns in various biological processes, and to determine mechanisms of drug action. The aim of this series of experiments was to gain experience in antipsychotic gene-expression profiling and characterize (in the parlance of genomics) the "antipsychotic transcriptome." In this prospective animal study, broad-scale gene expression profiles were characterized for brains of rats treated with antipsychotics and compared with those of sham controls. We used DNA microarrays containing 8000 sequences to measure the expression patterns of multiple genes in rat fronto-temporo-parietal cortex after intraperitoneal treatment with haloperidol or risperidone. A number of transcripts were differentially expressed between control and treated samples, of which only 36 and 89 were found to significantly differ in expression as a result of exposure to haloperidol or risperidone, respectively (P<0.05). Acutely, 13 genes were more highly expressed and 15 transcripts were found to be significantly less abundant, whereas chronically nine genes were up-regulated and none of them was repressed in haloperidol-treated cortices. Risperidone acutely induced 43 and repressed 46 genes, and chronically over-expressed 6 and down-regulated 11 transcripts. Selected genes were assayed by real-time PCR, then normalized to beta-actin. These assays confirmed the significance of the array results for all transcripts tested. Despite their differing receptor affinity and selectivity, our findings indicate that haloperidol and risperidone interfere with cell survival, neural plasticity, signal transduction, ionic homeostasis and metabolism in a similar manner.