Carlos Buesa

Mouse neuron navigator 1, a novel microtubule-associated protein involved in neuronal migration.

The development of the nervous system (NS) requires the coordinated migration of multiple waves of neurons and subsequent processes of neurite maturation, both involving selective guidance mechanisms. In Caenorhabditis elegans, unc-53 codes for a new multidomain protein involved in the directional migration of a subset of cells. We describe here the first functional characterization of the mouse homologue, mouse Neuron navigator 1 (mNAV1), whose expression is largely restricted to the NS during development. EGFP-mNAV1 associates with microtubules (MTs) plus ends present in the growth cone through a new microtubule-binding (MTB) domain. Moreover, its overexpression in transfected cells leads to MT bundling. The abolition of mNAV1 causes loss of directionality in the leading processes of pontine-migrating cells, providing evidence for a role of mNAV1 in mediating Netrin-1-induced directional migration.

Lysosome-associated membrane protein 1 (LAMP-1) in Alzheimer's disease.

Lysosome‐associated membrane protein 1 (LAMP‐1) is a glycoprotein highly expressed in lysosomal membranes. The present study was initiated to test LAMP‐1 mRNA and protein levels in post mortem frontal cortex (area 8) of Alzheimer’s disease (AD) stages I–IIA/B and stages V–VIC of Braak and Braak, compared with age‐matched controls. TaqMan PCR assays and Western blots demonstrated upregulation of LAMP‐1 mRNA and protein in the cerebral cortex in ADVC. In addition, immunohistochemical studies have shown increased LAMP‐1 immunoreactivity in neurones, and in glial cells surrounding senile plaques, in AD cases. Interestingly, LAMP‐1 immunoreactivity has little correlation with phosphorylated tau deposition and neurofibrillary tangles (NFTs), as neurones with NFTs were rarely LAMP‐1 immunoreactive. In contrast, LAMP‐1 expression was enhanced in neurones with granulovacuolar degeneration. Finally, LAMP‐1 occurred in microglia and multinucleated giant cells in one AD case in whom amyloid burden was cleared following βA‐peptide immunization. These findings support the participation of lysosomes in βA‐amyloid and, probably, in hyperphosphorylated tau turnover in AD.

Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies.

Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.

KDM1 histone lysine demethylases as targets for treatments of oncological and neurodegenerative disease

Histone methylation and demethylation are important processes associated with the regulation of gene transcription, and alterations in histone methylation status have been linked to a large number of human diseases. Initially thought to be an irreversible process, histone methylation is now known to be reversed by two families of proteins containing over 30 members that act to remove methyl groups from specific lysine residues present in the tails of histone H3 and histone H4. A rapidly growing number of reports have implicated the FAD-dependent lysine specific demethylase (KDM1) family in cancer, and several small-molecule inhibitors are in development for the treatment of cancer. An additional role has emerged for KDM1 in brain function, offering additional opportunities for the development of novel therapeutic strategies in neurodegenerative disease. A decade after the identification of KDM1A as a histone demethylase, the first selective inhibitors have now reached the clinic.

Coordinated Expression and Activity of 3- Hydroxy-3-Methylglutaryl Coenzyme A Synthase and Reductase in the Fat Body of Blattella germanica (L.) During Vitellogenesis

Levels of mRNA for the two 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthases, (HMG-S1 and HMG-S2), and for HMG-CoA reductase (HMG-R) of Blattella germanica were analyzed in the fat body during the first gonadotrophic cycle. HMG-S2 and HMG-R showed the highest mRNA levels on day 0 and decreased thereafter, whereas HMG-S1, showed faint expression. Western blot using specific antibodies for HMG-S1 and HMG-S2 showed no detectable levels for HMG-S1 but a clear pattern for HMG-S2. Both results point to a very limited role for HMG-CoA synthase-1 in B. germanica fat body that the functional enzyme in this organ is HMG-CoA synthase-2. HMG-CoA reductase and synthase proteins shared a cyclic pattern (maximum levels at day 4 and minimum levels on days 0 and 8), which was coincident with the pattern of activity. The delay between gene transcription and protein synthesis suggests a finely regulated translation mechanism. Moreover, the pattern of mevalonate synthesis parallels that of vitellogenin production, suggesting a coordinate mechanism between the mevalonate pathway and the production of vitellogenin.

Expression and activity of 3‐hydroxy‐3‐methylglutaryl‐CoA synthase and reductase in the fat body of ovariectomized and allatectomized Blattella germanica

Abstract. . 3‐Hydroxy‐3‐methylglutaryl‐CoA (HMG‐CoA) synthase and HMG‐CoA reductase show coordinated regulation in the fat body of Blattella germanica females. Since the profile of activity is parallel to the cycle of vitellogenin production, we postulated a link between the mevalonate pathway and vitellogenesis. Here we have studied both enzymes in females of B.germanica modified by ovariectomy (which leads to a saturable accumulation of vitellogenin) and allatectomy (which supresses vitellogenesis). Protein levels and enzymatic activity for both enzymes in ovariectomized specimens rose early in the first days of imaginal life and remained high until the end of the period studied, whereas controls showed cyclic profiles. In allatectomized specimens the same parameters were measured on day 4 of adult life and values were much lower with respect to controls. The parallelism between the patterns of HMG‐CoA synthase and reductase, and that of vitellogenin, suggests a functional relationship between the mevalonate pathway and the glycosylation of vitellogenin through dolichol intermediates.

Catalytic properties of recombinant 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase-1 from Blattella Germanica

Blattella germanica is the first organism in which two cytosolic HMG-CoA synthase genes have been described: HMGS-1 (Martínez-González et al., 1993b) and HMGS-2 (Buesa et al., 1994). The HMGS-1 gene showed special features, which led us to characterize the kinetic properties of the enzyme it encodes. Here we report the expression of recombinant HMGS-1, the protocol of enzyme purification, and the measurement of kinetic parameters. The K(m) for acetyl-CoA is 15.2 microM and the Ki for the other substrate, acetoacetyl-CoA, is 1.26 microM, both similar to that of yeast, ox, and chicken liver enzymes; the Vmax of HMGS-1 measured in this paper is 66 mU, which is the lowest Vmax of the HMG-CoA synthases reported to date.

The dual lsd1/maob inhibitor ory2001 prevents the development of the memory deficit in samp8 mice through induction of neuronal plasticity and reduction of neuroinflammation

P4-315 EVALUATING AMYLOID-BETA PROCESSING USING NOVEL GAMMA-SECRETASE MODULATORS IN PRECLINICAL ANIMAL MODELS Kathleen M. Wood, Cathleen Gonzales, Feng Pan, Nikolay Pozdnyakov, Michael Marconi, Ashley Robshaw, David Riddell, Dmitri Volfson, Martin Pettersson, Eva Hajos-Korcsok, Kelly R. Bales, Pfizer Inc., Cambridge, MA, USA; Merck, Cambridge, MA, USA; Merck, Indianapolis, IN, USA. Contact e-mail: kathleen.m. wood@pfizer.com