Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins.

Microglial interaction with amyloid fibrils in the brains of Alzheimer's and prion disease patients results in the inflammatory activation of these cells. We observed that primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar beta-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The tyrosine kinases Lyn and Syk are activated by the fibrillar peptides and initiate a signaling cascade resulting in a transient release of intracellular calcium that results in the activation of classical PKC and the recently described calcium-sensitive tyrosine kinase PYK2. Activation of the MAP kinases ERK1 and ERK2 follows as a subsequent downstream signaling event. We demonstrate that PYK2 is positioned downstream of Lyn, Syk, and PKC. PKC is a necessary intermediate required for ERK activation. Importantly, the signaling response elicited by beta-amyloid and prion fibrils leads to the production of neurotoxic products. We have demonstrated in a tissue culture model that conditioned media from beta-amyloid- and prion-stimulated microglia or from THP-1 monocytes are neurotoxic to mouse cortical neurons. This toxicity can be ameliorated by treating THP-1 cells with specific enzyme inhibitors that target various components of the signal transduction pathway linked to the inflammatory responses.

Phenotypic consequences of a nonsense mutation in the leptin receptor gene (fak) in obese spontaneously hypertensive Koletsky rats (SHROB).

The genetically obese Koletsky rat (SHROB, fak) has a novel point mutation of the leptin receptor at amino acid +763, resulting in a premature stop codon in the leptin receptor extracellular domain. This implies that all leptin receptor isoforms should be absent in this model. We examined the phenotypic consequences of this mutation on leptin and leptin receptor mRNA in hypothalamus and peripheral tissues from SHROB and their lean SHR littermates. Despite the mutation, mRNA for both the long (ObRa) and the short (ObRb) form were expressed at comparable levels in SHROB and SHR in brain and throughout peripheral tissues. Adipose tissue mRNA for leptin was two to three times greater in SHROB compared to SHR (P < 0.01), while circulating leptin concentration was 170 times greater than SHR littermates (P < 0.01), suggesting extreme leptin resistance in SHROB. Leptin was also detected in the cerebrospinal fluid (CSF) of SHR and SHROB (13.8 and 27.2 pmol/L, respectively); however, the CSF/plasma ratio for leptin was 32-fold greater in SHR than in SHROB. To assess the putative action of leptin and leptin receptors on insulin-mediated glucose transport, muscles from SHR and SHROB were incubated in vitro with recombinant human leptin. Leptin directly suppressed insulin-mediated glucose transport by 50% in skeletal muscle from SHR but not in obese SHROB rats lacking all forms of the leptin receptor. These results suggest that the natural leptin receptor knockout in the SHROB represents a unique rat model to define the functional role(s) of leptin in central and peripheral energy metabolism.

Cardiac and autonomic nervous system effects of L-alphacetylmethadol (LAAM).

The effects of 1-alphacetylmethadol (LAAM) on heart rate and force of contraction of isolated guinea-pig hearts and on release of tritium from sympathetic nerves were investigated. In vitro perfusion with LAAM depressed resting heart rate and right ventricular pressure. Nerve stimulation-induced tritium overflow was inhibited in a concentration related manner by LAAM. Increasing the calcium concentration in the perfusate partially inhibited the negative chronotropy and completely prevented the negative inotropic effect. One micromolar atropine prevented the negative chronotropy, partially inhibited the negative inotropic effect of LAAM and the depression of tritium release. Ten micromolar naltrexone failed to antagonize the effects of LAAM. Our results suggest that LAAM: 1) has a direct myocardial depressant effect which may be due to an inhibition of calcium influx; 2) produces a negative chronotropy through stimulation of atrial muscarinic receptors; and 3) interacts with presynaptic muscarinic receptors which modulate nerve stimulation-induced release of noradrenaline.