Marco Racchi

Dysfunction of the cholesterol biosynthetic pathway in Huntington's disease.

The expansion of a polyglutamine tract in the ubiquitously expressed huntingtin protein causes Huntingtons disease (HD), a dominantly inherited neurodegenerative disease. We show that the activity of the cholesterol biosynthetic pathway is altered in HD. In particular, the transcription of key genes of the cholesterol biosynthetic pathway is severely affected in vivo in brain tissue from HD mice and in human postmortem striatal and cortical tissue; this molecular dysfunction is biologically relevant because cholesterol biosynthesis is reduced in cultured human HD cells, and total cholesterol mass is significantly decreased in the CNS of HD mice and in brain-derived ST14A cells in which the expression of mutant huntingtin has been turned on. The transcription of the genes of the cholesterol biosynthetic pathway is regulated via the activity of sterol regulatory element-binding proteins (SREBPs), and we found an ∼50% reduction in the amount of the active nuclear form of SREBP in HD cells and mouse brain tissue. As a consequence, mutant huntingtin reduces the transactivation of an SRE-luciferase construct even under conditions of SREBP overexpression or in the presence of an exogenous N-terminal active form of SREBP. Finally, the addition of exogenous cholesterol to striatal neurons expressing mutant huntingtin prevents their death in a dose-dependent manner. We conclude that the cholesterol biosynthetic pathway is impaired in HD cells, mice, and human subjects, and that the search for HD therapies should also consider cholesterol levels as both a potential target and disease biomarker.

Insulin regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase-dependent pathway

Several lines of biochemical evidence correlate the presence of energy metabolic defects with the functional alterations associated with brain aging and with the pathogenesis of neurodegenerative disorders such as Alzheimers disease. Within this context we tested the ability of insulin to regulate the amyloid precursor protein (APP) processing in SH‐SY5Y neuroblastoma cells. Our findings show that insulin promotes APP metabolism by a glucose‐independent mechanism. We demonstrate a novel intracellular pathway that increases the rate of secretion of soluble APP through the activity of phosphati‐dyl‐inositol 3 kinase (PI3‐K). This pathway, downstream of insulin receptor tyrosine kinase activity, does not involve either the activation of protein kinase C or the mitogen‐activated protein kinase (MAP‐K) pathway. Because of the physiological role of PI3‐K in the translocation of glucose transporter‐containing vesicles, we speculate that PI3‐K involvement in APP metabolism may act at the level of vesicular trafficking.—Solano, D. C., Sironi, M., Bon‐fini, C., Solerte, S. B., Govoni, S., Racchi, M. Insulin regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase‐dependent pathway. FASEBJ. 14, 1015–1022 (2000)

Cytosol protein kinase C downregulation in fibroblasts from Alzheimer's disease patients

We attempted to determine whether changes in protein kinase C (PKC) activity in Alzheimers disease (AD) brains are also present in cultured skin fibroblasts from living patients. Biopsies collected from shoulder skin were transferred to culture plates with an appropriate growth medium, and histone-directed PKC activity as well as phorbol ester binding were individually determined in soluble and particulate fractions prepared from AD and non-AD fibroblast cell lines. Binding experiments indicated that PKC was unevenly distributed between cytosol (78%) and particulate (22%). The Bmax values for phorbol ester binding in soluble and particulate fractions were similar in AD and non-AD patients. Kd values in the cytosol were 94% higher in AD patients, indicating lower affinity of the enzyme for the ligand. Accordingly, the soluble PKC activity was 30% lower in AD patients. The data suggest that the changes in PKC phosphorylating activity represent a diffuse cellular defect in AD and are not confined to the brain. The alterations of the enzyme may participate in the disregulation in processing of β-amyloid precursor protein in AD.

Activation of the Notch pathway in Down syndrome: cross-talk of Notch and APP

Down syndrome (DS) patients suffer from mental retardation, but also display enhanced β‐APP production and develop cortical amyloid plaques at an early age. As β‐APP and Notch are both processed by γ‐secretase, we analyzed expression of the Notch signaling pathway in the adult DS brain and in a model system for DS, human trisomy 21 fibroblasts by quantitative PCR. In adult DS cortex we found that Notch1, Dll1 and Hes1 expression is up‐regulated. Moreover, DS fibroblasts and Alzheimer disease cortex also show overexpression of Notch1 and Dll1, indicating that enhanced β‐APP processing found in both DS and AD could be instrumental in these changes. Using pull‐down studies we could demonstrate interaction of APP with Notch1, suggesting that these transmembrane proteins form heterodimers, but independent of γ‐secretase. We could demonstrate binding of the intracellular domain of Notch1 to the APP adaptor protein Fe65. Furthermore, activated Notch1 can trans‐activate an APP target gene, Kai1, and vice versa, activated APP can trans‐activate the classical Notch target gene Hes1. These data suggest that Notch expression is activated in Down syndrome, possibly through cross‐talk with APP signaling. This interaction might affect brain development, since the Notch pathway plays a pivotal role in neuron‐glia differentiation.—Activation of the Notch pathway in Down syndrome: cross‐talk of Notch and APP. Fischer D. F., Van Dijk R., Sluijs J. A., Nair S. M., Racchi M., Levelt C. N., Van Leeuwen F. W., Hol E. M. Activation of the Notch pathway in Down syndrome: cross‐talk of Notch and APP. FASEB J. 19, 1451–1458 (2005)

Calcium responses in human fibroblasts: A diagnostic molecular profile for Alzheimer's disease

We have previously identified alterations of K+ channel function, IP3-mediated calcium release, and Cp20 (a memory-associated GTP binding protein) in fibroblasts from AD patients vs. controls. In the present study we introduce a scoring system based on these response alterations that integrates two or more alterations (and their degree) in AD vs. control fibroblasts. This scoring system generates an index that distinguishes AD patients from controls with both high specificity and sensitivity. We also show that low doses of bradykinin elicit intracellular calcium release almost exclusively in AD cell lines in an all or none fashion that provide a clear measurement of enhanced IP3-mediated function in AD vs. controls.

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells

The present study investigates the influence of energy related metabolic stress on amyloid precursor protein (APP) non-amyloidogenic secretory processing in COS cells. The effect of glucose deprivation on soluble APP (sAPP) secretion has been evaluated: incubation of COS cells with 50 mM 2-deoxy-D-glucose (2-DG) in glucose free medium was able to reduce sAPP secretion (-26%). Sodium azide (NaN3), an inhibitor of cytochrome c oxidase (complex IV of the mitochondrial electron transfer chain) decreased sAPP release in a concentration dependent way (maximum -75%). Treatment of COS cells with the antioxidant glutathione (GSH) fully antagonized the inhibitory effect of azide (1 mM) and elicited sAPP release over basal level. These results suggest that the inhibition of energy metabolism can influence APP processing leading to a decreased secretion of non-amyloidogenic fragments of APP.

p53 at the crossroads between cancer and neurodegeneration.

Aging, dementia, and cancer share a critical set of altered cellular functions in response to DNA damage, genotoxic stress, and other insults. Recent data suggest that the molecular machinery involved in maintaining neural function in neurodegenerative disease may be shared with oncogenic pathways. Cancer and neurodegenerative diseases may be influenced by common signaling pathways regulating the balance of cell survival versus death, a decision often governed by checkpoint proteins. This paper focuses on one such protein, p53, which represents one of the most extensively studied proteins because of its role in cancer prevention and which, furthermore, has been recently shown to be involved in aging and Alzheimer disease (AD). The contribution of a conformational change in p53 to aging and neurodegenerative processes has yet to be elucidated. In this review we discuss the multiple functions of p53 and how these correlate between cancer and neurodegeneration, focusing on various factors that may have a role in regulating p53 activity. The observation that aging and AD interfere with proteins controlling duplication and cell cycle may lead to the speculation that, in senescent neurons, aberrations in proteins generally dealing with cell cycle control and apoptosis could affect neuronal plasticity and functioning rather than cell duplication.

Oxidative metabolism in cultured fibroblasts derived from sporadic Alzheimer's disease (AD) patients

Fibroblasts from Alzheimers disease (AD) patients displayed decreased cytochrome c oxidase (complex IV) activity (P < 0.05). The basal oxygen consumption rate (QO2) and the response to an uncoupler of oxidative phosphorylation did not differ between AD and control fibroblasts. The QO2 of AD fibroblasts was more susceptible (P < 0.05) to inhibition by azide in the range 0.5-5 mM. The basal intracellular pH (pHi) in AD fibroblasts was significantly more acidic than in control ones. The results support the hypothesis that subtle dysfunctions of oxidative energy-producing processes are present in fibroblasts from sporadic AD patients. The alterations observed scantly influence the fibroblasts functioning even in stressful conditions; however in tissues, such as the brain, that rely heavily on oxidative metabolism for their function, similar alterations may trigger molecular mechanisms leading to cell damage.

Phosphorylation of APP-CTF-AICD domains and interaction with adaptor proteins: signal transduction and/or transcriptional role – relevance for Alzheimer pathology

J. Neurochem. (2010) 115, 1299–1308.

Specific role for protein kinase Cα in the constitutive and regulated secretion of amyloid precursor protein in human skin fibroblasts

Reduced levels of protein kinase C alpha (PKC alpha) seems to be related to an altered amyloid precursor protein (APP) secretion in fibroblasts from Alzheimers disease (AD) patients. In this report we used a specific inhibitor of PKC alpha (Gö-6976), to investigate the role of PKC alpha in the basal and phorbol esters regulated secretion of soluble APP (sAPP) in human fibroblasts derived from healthy aged volunteers. Treatment with Gö-6976 alone reduced basal secretion by a maximum of 39%, compared to untreated cells, suggesting the partial dependence of constitutive APP secretory pathway on PKC alpha enzyme. Moreover Gö-6976 treatment completely abolished the effect of phorbol-esters mediated PKC stimulation on sAPP release, suggesting that PKC alpha is the only PKC isoform involved in controlling the secretion of sAPP in human fibroblasts.