LRRK2 and the stress response: interaction with MKKs and JNK-interacting proteins.

Increasing evidence supports a putative link between LRRK2 function and the MAP kinase cascades. We recently demonstrated that LRRK2 binds to MKK6, -3, and -7. Previous studies demonstrated that scaffold proteins are essential in the regulation of subcellular localization of stress kinase complexes. The c-jun NH2-terminal kinase (JNK)-interacting proteins (JIPs) are a group of scaffold proteins that play an important role in the regulation of MAP kinase signaling cascades. JIP1-3 are known to regulate the specificity and localization of the JNK pathway, while JIP4 is a specific scaffolding protein for the p38 pathway. We demonstrate that LRRK2 binds to JIP1-4, and is associated with increased levels of total JIP1, -3, -4, oligomeric JIP and ubiquitinated JIP. These results are consistent with a putative role of LRRK2 in regulating the stress kinase cascade.

Lipid-lowering treatment is related to decreased risk of dementia: a population-based study (FINRISK).

BACKGROUND: Several lines of evidence have linked cholesterol to dementia. OBJECTIVE: To investigate lipid-lowering drug use and dementia development in a Finnish population. METHODS: FINRISK is a large population-based survey of cardiovascular risk factors carried out since 1972 every 5 years using independent, random and representative population samples from different parts of Finland. Several cohorts were part of the WHO-MONICA study. Data from cohorts 1972-2002 were linked to the Hospital Discharge Registry and Drug Reimbursement Registry (1995-2007) to ascertain dementia diagnoses and lipid-lowering treatment. Selection criteria for the study were: (1) alive and without dementia in 1995; (2) age > or = 60 years (in 1995 for earlier cohorts and in 1997 or 2002 for later cohorts; (3) treatment prescribed at least 1 year before dementia diagnosis. RESULTS: 17,597 persons were included in the study. Lipid-lowering treatment was related to decreased dementia risk. In Cox proportional hazards model, hazard ratio (95% CI) was 0.42 (0.37-0.49; controlled for age, sex, education, survey region, survey year, baseline cholesterol, body mass index and systolic blood pressure). CONCLUSION: Preliminary results from the FINRISK study indicate that lipid-lowering drugs may have a beneficial effect in dementia prevention. Further data linkage is ongoing in order to investigate the roles of different types of lipid-lowering drugs.

A neuronal antigen in the brains of Alzheimer patients.

A monoclonal antibody was prepared against pooled homogenates of brain tissue from patients with Alzheimer's disease. This antibody recognizes an antigen present in much higher concentration in certain brain regions of Alzheimer patients than in normal brain. The antigen appears to be a protein present in neurons involved in the formation of neuritic plaques and neurofibrillary tangles, and in some morphologically normal neurons in sections from Alzheimer brains. Partial purification and Western blot analysis revealed the antigen from Alzheimer brain to be a single protein with a molecular weight of 68,000. Application of the same purification procedure to normal brain tissue results in the detection of small amounts of a protein of lower molecular weight.

Participation of presenilin 2 in apoptosis: enhanced basal activity conferred by an Alzheimer mutation.

Overexpression of the familial Alzheimer's disease gene Presenilin 2 (PS2) in nerve growth factor-differentiated PC12 cells increased apoptosis induced by trophic factor withdrawal or beta-amyloid. Transfection of antisense PS2 conferred protection against apoptosis induced by trophic withdrawal in nerve growth factor-differentiated or amyloid precursor protein-expressing PC12 cells. The apoptotic cell death induced by PS2 protein was sensitive to pertussis toxin, suggesting that heterotrimeric GTP-binding proteins are involved. A PS2 mutation associated with familial Alzheimer's disease was found to generate a molecule with enhanced basal apoptotic activity. This gain of function might accelerate the process of neurodegeneration that occurs in Alzheimer's disease, leading to the earlier age of onset characteristic of familial Alzheimer's disease.

Re-assessing the relationship between cholesterol, statins and Alzheimer's disease.

This communication integrates the purported role of cholesterol and statins in Alzheimer's disease (AD) with recent data. Meta-analysis of association studies relevant to AD indicates that apolipoprotein (apo)E4 is the only cholesterol-related polymorphism that shows clear association with AD. This suggests that the effect of apoE4 on the pathophysiology of AD occurs via a mechanism that is not directly related to cholesterol, such as fibrillization of Abeta. Despite the lack of genetic association, cholesterol and statins clearly modulate amyloid precursor protein (APP) processing in cell culture and animal models. Statins appear to act by a pleiotropic mechanism, involving both cholesterol (via lipid rafts) and isoprenylation. The pleiotropic mechanism of statin action clarifies conflicting data from clinical studies, where statins exert an action on Abeta and AD that might be dose dependent because of actions on both cholesterol and isoprenylation. Reduced isoprenylation can also inhibit inflammation. Our own studies of brains from Alzheimer subjects +/- statins indicate that statins inhibit inflammation in humans but might not reduce cerebral Abeta load. These results suggest that the primary action of statins in humans with AD might be to reduce inflammation rather than decrease Abeta load.

alpha-Synuclein shares physical and functional homology with 14-3-3 proteins.

alpha-Synuclein has been implicated in the pathophysiology of many neurodegenerative diseases, including Parkinson's disease (PD) and Alzheimer's disease. Mutations in alpha-synuclein cause some cases of familial PD (Polymeropoulos et al., 1997; Kruger et al., 1998). In addition, many neurodegenerative diseases show accumulation of alpha-synuclein in dystrophic neurites and in Lewy bodies (Spillantini et al., 1998). Here, we show that alpha-synuclein shares physical and functional homology with 14-3-3 proteins, which are a family of ubiquitous cytoplasmic chaperones. Regions of alpha-synuclein and 14-3-3 proteins share over 40% homology. In addition, alpha-synuclein binds to 14-3-3 proteins, as well as some proteins known to associate with 14-3-3, including protein kinase C, BAD, and extracellular regulated kinase, but not Raf-1. We also show that overexpression of alpha-synuclein inhibits protein kinase C activity. The association of alpha-synuclein with BAD and inhibition of protein kinase C suggests that increased expression of alpha-synuclein could be harmful. Consistent with this hypothesis, we observed that overexpression of wild-type alpha-synuclein is toxic, and overexpression of alpha-synuclein containing the A53T or A30P mutations exhibits even greater toxicity. The activity and binding profile of alpha-synuclein suggests that it might act as a protein chaperone and that accumulation of alpha-synuclein could contribute to cell death in neurodegenerative diseases.

Specific chemical modification of the readily nitrated tyrosine of the RTEM beta-lactamase and of bacillus cereus beta-lactamase I. The role of the tyrosine in beta-lactamase catalysis.

The function of the hydroxyl group of the tyrosine residue readily nitrated by tetranitromethane (tyrosine-105) in the RTEM plasmid-derived beta-lactamase (penicillinase; penicillin amido beta-lactam-hydrolase, EC 3.5.1.6) from E. coli and in Bacillus cereus beta-lactamase I has been investigated by chemical modification methods. In the case of B. cereus beta-lactamase I the nitrated tyrosine can be acetylated by acetic anhydride without effect on beta-lactamase activity The nitrated tyrosine of the E. coli enzyme can also be acetylated but in this case beta-lactamase activity is lost in a manner which directly correlates with extent of acetylation. However, deacetylation of the nitrotyrosine does not restore activity. The dilemma created by the latter result has been resolved by development of a new method of tyrosine hydroxyl modification at low pH. The nitrated enzyme is reduced by dithionite and then treated with either carbonyldiimidazole or N-(2.2.2-trifluoroethoxycarbonyl)imidazole, both of which convert 3-aminotyrosine into benzoxazolinonylalanine. That the final modification has been achieved is demonstrated both by classical chemical methods and by employment of Fourier transform infrared spectroscopy to detect the characteristic benzoxazolinone carbonyl absorption. Further, it is shown that no significant loss of beta-lactamase activity is associated with this modification. Hence in neither the B. cereus or the E. coli enzyme does the readily nitrated tyrosine residue have a direct chemical function at the beta-lactamase active site.

A.E. Bennett Research Award 1993. Olfactory neuroblasts from Alzheimer donors: studies on APP processing and cell regulation.

Cell lines of continuously dividing human olfactory neuroblasts can be propagated using olfactory epithelium obtained from human donors at biopsy or autopsy. The expression of neuronal proteins in these cells, such as neurofilament protein and tau protein, can be increased using a combination of factors including nerve growth factor, fibroblast growth factor, interleukin 1 and interleukin 6. These cells also express aspects of human disease. Olfactory neuroblasts generated from donors with the common, sporadic forms of Alzheimer's disease, show elevated levels of the direct precursor to beta-amyloid, the amyloid precursor protein C-terminal derivative (CTD). When treated with the lysosomal inhibitor chloroquine, immunoblots of Alzheimer olfactory neuroblasts show seven-fold higher levels of CTDs than immunoblots from age-matched control neuroblasts. The disease related increases in CTDs can be reversed by treatment with agents that increase intracellular cyclic adenosine monophosphate (cAMP), such as dibutyryl-cyclic-AMP, theophylline, and isoproterenol.

Primary structure of the serotonin transporter in unipolar depression and bipolar disorder.

Genetic factors have been implicated in the etiology of affective disorders but due to the complex inheritance patterns of these disorders, identification of the responsible gene(s) has so far been unsuccessful. Decreased platelet serotonin (5-HT) transport and reduced binding of imipramine or paroxetine to brain and platelet 5-HT uptake sites/transporters in patients with depression and suicide victims define the 5-HT transporter (5-HTT) as a candidate gene. The primary structure of the 5-HTT was analyzed in 17 patients meeting DSM-III-R diagnostic criteria for major depressive or bipolar disorder and in 4 healthy controls using polymerase chain reaction (PCR-) amplification and sequencing of complementary deoxyribose nucleic acid (cDNA) synthesized from platelet 5-HTT messenger ribose nucleic acid (mRNA). Direct PCR sequencing of the protein coding region failed to reveal changes in the deduced amino acid sequence of the platelet/brain 5-HTT (40,000 base pairs sequence screened), although a conservative single-base substitution representing a silent polymorphism was found. The results provide preliminary evidence that alterations in the primary structure of 5-HTT are not generally involved in the pathogenesis of unipolar depression and manic-depressive illness.

Longitudinal stability of CSF tau levels in Alzheimer patients.

BACKGROUND: Antemortem levels of tau in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) patients have repeatedly been demonstrated to be elevated when compared to controls. Although CSF tau has been reported to be elevated even in very mild AD, it is unknown how tau levels change during the course of the disease. METHODS: We have followed 29 mild-to-moderately affected AD subjects over 2 years with repeated CSF taps. Clinical measures of dementia severity (Clinical Dementia Rating Scale, Global Deterioration Scale and Mini-Mental Status Examination) were obtained at the start and conclusion of the observation period, and CSF tau was measured with a standard enzyme-linked immunoabsorbent assay (ELISA) using two monoclonal antibodies. RESULTS: Despite significant changes in the clinical measures consistent with progression of the disease, no significant overall change in CSF tau levels (548 +/- 355 vs. 557 +/- 275 pg/mL, NS) was observed. None of the clinical variables was significantly correlated with either baseline measures of CSF tau or delta CSF tau (last-first). Similarly, CSF tau at baseline and changes over time were not significantly related to Apolipoprotein E (APO E) phenotype. CONCLUSIONS: These data suggest that CSF tau levels are stable over extended periods of time in a group of mild-to-moderately demented AD subjects and that CSF tau levels do not predict the severity or rate of progression of AD, at least not during the middle stages of the illness.

Regulation of apoptosis by presenilin 1.

Familial Alzheimer's disease is transmitted as an autosomal dominant disorder and, in 5-10% of the cases, is caused by mutations in the coding regions of two homologous genes, Presenilin 1 and 2 (PS1 and PS2). Previously, we have shown that PS2, a homolog of PS1. regulates apoptosis induced in neurons by trophic withdrawal or Abeta, and in T-cells by Fas ligand. We now report that PS1 also regulates apoptosis. Both wild-type and the H115Y mutant form of PS1 enhance Fas-mediated apoptosis in Jurkat cells. We also observed that wild-type and the H115Y mutant form of PS1 differentially regulate Jun Kinase, an important enzyme regulating apoptosis.

Protein alterations in olfactory neuroblasts from Alzheimer donors.

Definitive diagnosis of Alzheimer's disease (AD) is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. To date, there are no good biological markers for a positive diagnosis of AD in the living patient. In an effort to identify biological markers useful both in the clinical and pathologic diagnosis of AD, we have investigated disease-specific protein alterations in cultured olfactory neurons. Olfactory neurons are readily accessible by biopsy, can be propagated in primary cell culture as olfactory neuroblasts (ONs), and exhibit several elements of AD brain pathophysiology making them powerful tools for the study of AD. Two-dimensional gel analysis of ON proteins from neuropsychologically evaluated AD donors revealed a set of five proteins (Mr 17-50 kD, pI 4.8-6.7) that were significantly altered in concentration when compared to cells from age-matched controls. Further characterization and microsequence analysis could lead to the identification of proteins that may have important diagnostic or therapeutic value in the treatment of AD.

Direct association of presenilin-1 with beta-catenin.

Families bearing mutations in the presenilin-1 (PSI) gene develop Alzheimer's disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co-immunoprecipitation with PS1 in transfected COS-7 cells indicates that PSI directly interacts with endogenous beta-catenin, and the interaction requires residues 322450 of PSI and 445-676 of beta-catenin. Both proteins are co-localized in the endoplasmic reticulum. Over-expression of PS1 reduces the level of cytoplasmic beta-catenin, and inhibits beta-catenin-T cell factor-regulated transcription. These results indicate that PSI plays a role as inhibitor of the beta-catenin signal, which may be connected with the AD dysfunction.

Decreased prevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors.

CONTEXT: Increasing evidence suggests that cholesterol plays a role in the pathophysiology of Alzheimer disease (AD). For instance, an elevated serum cholesterol level has been shown to be a risk factor for AD. OBJECTIVE: To determine whether patients taking 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), which are a group of medicines that inhibit the synthesis of cholesterol, have a lower prevalence of probable AD. DESIGN: The experiment uses a cross-sectional analysis comparing the prevalence of probable AD in 3 groups of patients from hospital records: the entire population, patients receiving 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (hereafter referred to as the statins), and patients receiving medications used to treat hypertension or cardiovascular disease. PATIENTS: The subjects studied were those included in the computer databases of 3 different hospitals for the years October 1, 1996, through August 31, 1998. MAIN OUTCOME MEASURES: Diagnosis of probable AD. RESULTS: We find that the prevalence of probable AD in the cohort taking statins during the study interval is 60% to 73% (P < .001) lower than the total patient population or compared with patients taking other medications typically used in the treatment of hypertension or cardiovascular disease. CONCLUSIONS: There is a lower prevalence of diagnosed probable AD in patients taking 2 different 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors-lovastatin and pravastatin. While one cannot infer causative mechanisms based on these data, this study reveals an interesting association in the data, which warrants further study. Arch Neurol. 2000;57:1439-1443

Continuous culture of neuronal cells from adult human olfactory epithelium.

Cells from the olfactory epithelium of adult human cadavers have been propagated in primary culture and subsequently cloned. These cells exhibit neuronal properties including: neuron-specific enolase, olfactory marker protein, neurofilaments, and growth-associated protein 43. Simultaneously, the cells exhibit nonneuronal properties such as glial fibrillary acidic protein and keratin, the latter suggesting properties of neuroblasts or stem cells. These clonal cultures contain 5-10% of cells sufficiently differentiated to show odorant-dependent cyclic adenosine 3',5'-monophosphate (cAMP) or calcium-release responses when challenged with submicromolar concentrations of odorants. The potential of culturing neuronal cells from patients with neuropsychiatric disorders, such as Alzheimer's disease or schizophrenia, could enable the study of the pathophysiology of these neurons in the culture dish and allow new approaches to the study of mental illness.

FMR1 gene expression in olfactory neuroblasts from two males with fragile X syndrome.

The fragile X mental retardation 1 gene (FMR1) mutation is strongly correlated with specific and marked neurobehavioral and neuroanatomical abnormalities. The protein product, FMRP, is highly expressed in neurons of the normal mammalian brain, and absent or in low levels in leukocytes from individuals with fragile X (FraX)-associated mental impairment. Inferences which arise from these findings are that FMRP has a critical role in the development and functioning of the brain, and that leukocyte-derived molecular assessments provide a good indicator of FMR1 expression in that organ. This latter conclusion appears true in most cases even though the typical FMR1 mutation is an unstable triplet repeat expansion which demonstrates somatic heterogeneity within and across tissues. Blood to brain correspondence in FraX patients has only rarely been confirmed by the direct study of human brain specimens and, to our knowledge, it has never been studied in living individuals with the FMR1 mutation. In this report, we describe the FMR1 patterns in olfactory neuroblasts (ON) from two living brothers with expansion mutations in their leukocytes who are mentally retarded and autistic. ON were chosen for study because they are accessible neurons closely linked to the brain. In both subjects, the ON genotype was highly, but not perfectly, consistent with that observed in leukocytes. Protein phenotypes across tissues were completely consistent showing the absence of FMRP-immunoreactivity (-ir). These results augment the limited amount of direct evidence which indicates that FMR1 mutation patterns in leukocytes are a good, albeit potentially fallible, reflection of such patterns in the brain. This report further demonstrates the feasibility of using ON samples to evaluate the FMR1 mutation in humans in vivo.

Recent advances in the neurochemistry of Alzheimer's disease.

This article reviews recent progress in research in the genesis of neurochemical changes in the Alzheimer brain, which has been primarily in two areas: the cellular proteins associated with the pathologic structures and the neurotransmitter changes. Research and clinical implications of recent studies and of works in progress are provided.

Amyloid precursor protein requires the insulin signaling pathway for neurotrophic activity.

Picomolar concentrations of purified amyloid precursor protein (APP) potentiate the neurotrophic activity of suboptimal concentrations of NGF on PC12 cells. To understand the molecular basis for this potentiation, we have characterized the signal transduction pathway used by APP for its neurotrophic activity. APP stimulated the tyrosine phosphorylation of a number of proteins including insulin receptor substrate-1 (IRS-1). Incubation of naive cells with antisense oligonucleotides to IRS-1 mRNA resulted in a dramatic reduction of IRS-1 levels and inhibition of APP stimulated neurite outgrowth. Phosphotidylinositol 3-kinase became associated with IRS-1 and activated upon APP stimulation. Extracellular signal-regulated kinase (ERK 1 and ERK 2) phosphorylation was detected by both immunoblot analysis and immunocytochemistry using antibodies directed to their phosphorylated (and hence, activated) form. There was also an elevation of ERK kinase activity. The potentiation of NGF activity was reflected in a correspondingly synergistic elevation of tyrosine phosphorylated ERK. The pattern of signal transduction targets indicates that APP potentiated the neurotrophic effects of NGF via the activation of the IRS-1 signaling pathway.

Regional brain expression of serotonin transporter mRNA and its regulation by reuptake inhibiting antidepressants.

Regional expression and antidepressant drug-induced regulation of mRNA encoding the serotonin (5-HT) transporter were studied in rat brain. While 5-HT transporter mRNA is abundantly expressed in the midbrain raphe complex, lower concentrations were also found in frontal cortex, hippocampus, and neostriatum using a combination of reverse transcriptase-polymerase chain reaction (RT-PCR), Southern hybridization, and sequence analysis. Long-term administration of antidepressants which inhibit 5-HT reuptake, but not monoamine oxidase inhibitors or 5-HT receptor agonists, decrease 5-HT transporter mRNA steady-state concentrations. Based on these observations, we conclude that (1) mRNA coding for the 5-HT transporter is present in several brain areas associated with ascending HT pathways, and (2) chronic treatment with reuptake inhibiting antidepressants may be associated with regulation of the 5-HT transporter at the level of gene expression which may contribute to the neuroadaptive mechanisms that likely underlie their therapeutic efficacy.

SEPT5_v2 is a parkin-binding protein.

Mutations in parkin are associated with various inherited forms of Parkinson's disease (PD). Parkin is a ubiquitin ligase enzyme that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins destined for proteasomal degradation. The substrates of parkin-mediated ubiquitination have yet to be completely identified. Using a yeast two-hybrid screen, we isolated the septin, human SEPT5_v2 (also known as cell division control-related protein 2), as a putative parkin-binding protein. SEPT5_v2 is highly homologous to another septin, SEPT5, which was recently identified as a target for parkin-mediated ubiquitination. SEPT5_v2 binds to parkin at the amino terminus and in the ring finger domains. Several lines of evidence have validated the putative link between parkin and SEPT5_v2. Parkin co-precipitates with SEPT5_v2 from human substantia nigra lysates. Parkin ubiquitinates SEPT5_v2 in vitro, and both SEPT5_v1 and SEPT5_v2 accumulate in brains of patients with ARJP, suggesting that parkin is essential for the normal metabolism of these proteins. These findings suggest that an important relationship exists between parkin and septins.