A comparative analysis of leucine-rich repeat kinase 2 (Lrrk2) expression in mouse brain and Lewy body disease.

Pathogenic substitutions in leucine-rich repeat kinase 2 (LRRK2, Lrrk2) have been genetically linked to familial, late-onset Parkinsonism. End-stage disease is predominantly associated with nigral neuronal loss and Lewy body pathology, but patients may have gliosis, tau or ubiquitin inclusions (pleomorphic pathology). The anatomical distribution of Lrrk2 protein may provide insight into its function in health and neurodegeneration, thus we performed a comparative study with 'in-house' and commercially available Lrrk2 antibodies using brain tissue from wild type and human Lrrk2 transgenic bacterial artificial chromosome (BAC) mice, and from diffuse Lewy body disease (DLBD) patients. Lrrk2 protein was ubiquitously expressed and relatively abundant in most brain regions, including the substantia nigra, thalamus and striatum. Lrrk2 was not a major component of Lewy body or neuritic pathology associated with Parkinson's disease. However, selective loss of dopaminergic neurons in Lrrk2-associated Parkinsonism argues the protein may have regional-specific interactions. Lrrk2 immunohistochemical staining was present in the subventricular zone, a region containing stem cells that give rise to both neurons and glia. A role for Lrrk2 in neurogenesis might provide further insight into the aberrant role of mutant protein in age-associated neurodegeneration with pleomorphic pathology.

PKA phosphorylations on tau: developmental studies in the mouse.

PKA phosphorylations of tau may be an early event in the development of neurofibrillary pathology in Alzheimer's disease. Serines 214 and 409 of tau are highly phosphorylated in PHF-tau, but are not phosphorylated to any significant extent in normal adult human brain; both of these sites are phosphorylated in human fetal tissue. To further study this phenomenon, a developmental characterization of these phosphorylation sites relative to PKA, cAMP-dependent response binding element (CREB) and phosphorylated CREB was performed using samples from mouse brain. Immunoblot analysis using antibodies specific for phospho-serine 214 (CP-3) and phospho-serine 409 (PG-5) revealed a marked decrease in phosphorylation occurring at each of these sites between postnatal day 11 (P11) and P20. Immunoblots with TG-5, a pan-tau antibody, revealed uniform expression of tau during postnatal development, as well as a switch in isoform composition that is evident between P7 and P11. This switch in isoform composition just precedes the change in the extent of phosphorylation at serines 214 and 409, and occurs at a time when PKA phosphorylation of CREB is increasing.

Isolation and characterization of cDNA clones encoding antifreeze proteins of the pyrochroid beetle Dendroides canadensis.

Temporal differences in the expression of Dendroides canadensis antifreeze protein (DAFP) are indicated from seasonal comparison of dafp-1 transcript level, thermal hysteresis activity and temperature changes. DAFP-1 transcript abundance correlates with the thermal hysteresis activity level in late fall/early winter and appears to follow overall seasonal temperature changes with peak transcript levels occurring in December. A cDNA library created from December larvae yielded clones encoding a set of novel putative DAFPs. Some of the cDNA clones isolated display significant divergence at the primary amino acid level, yet, maintain conservation of key residues that are presumably important for structure and function of antifreeze proteins in this cold-hardy organism. Seasonal analysis of two dafps (dafp-1 and dafp-7) revealed differences on the transcriptional level, suggesting that DAFPs may serve somewhat different functions.

Enhancement of insect antifreeze protein activity by solutes of low molecular mass.

Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ice) below the melting point, thereby producing a difference between the freezing and melting points that has been termed thermal hysteresis. In general, the magnitude of the thermal hysteresis depends upon the specific activity and concentration of the AFP. This study describes several low-molecular-mass solutes that enhance the thermal hysteresis activity of an AFP from overwintering larvae of the beetle Dendroides canadensis. The most active of these is citrate, which increases the thermal hysteresis nearly sixfold from 1.2 degrees C in its absence to 6.8 degrees C. Solutes which increase activity approximately fourfold are succinate, malate, aspartate, glutamate and ammonium sulfate. Glycerol, sorbitol, alanine and ammonium bicarbonate increased thermal hysteresis approximately threefold. Interestingly, 0.5 mol l-1 sodium sulfate eliminated activity. Solute concentrations between 0.25 and 1 mol l-1 were generally required to elicit optimal thermal hysteresis activity. Glycerol is the only one of these enhancing solutes that is known to be present at these concentrations in overwintering D. canadensis, and therefore the physiological significance of most of these enhancers is unknown. The mechanism(s) of this enhancement is also unknown. The AFP used in this study (DAFP-4) is nearly identical to previously described D. canadensis AFPs. The mature protein consists of 71 amino acid residues arranged in six 12- or 13-mer repeats with a consensus sequence consisting of Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend to be charged residues, X5 tends to be Thr or Ser, X6 to be Asn or Asp, X9 to be Asn or Lys and X13 to be Ala in the 13-mers. DAFP-4 is shorter by one repeat than previously described D. canadensis AFPs.

Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis.

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend toward charged residues, X5 tends toward threonine or serine, X6 toward asparagine or aspartate, X9 toward asparagine or lysine, and X13 toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor.