Expression Profiling of a Genetic Animal Model of Depression Reveals Novel Molecular Pathways Underlying Depressive-Like Behaviours

Background

The Flinders model is a validated genetic rat model of depression that exhibits a number of behavioural, neurochemical and pharmacological features consistent with those observed in human depression.

Principal Findings

In this study we have used genome-wide microarray expression profiling of the hippocampus and prefrontal/frontal cortex of Flinders Depression Sensitive (FSL) and control Flinders Depression Resistant (FRL) lines to understand molecular basis for the differences between the two lines. We profiled two independent cohorts of Flinders animals derived from the same colony six months apart, each cohort statistically powered to allow independent as well as combined analysis. Using this approach, we were able to validate using real-time-PCR a core set of gene expression differences that showed statistical significance in each of the temporally distinct cohorts, representing consistently maintained features of the model. Small but statistically significant increases were confirmed for cholinergic (chrm2, chrna7) and serotonergic receptors (Htr1a, Htr2a) in FSL rats consistent with known neurochemical changes in the model. Much larger gene changes were validated in a number of novel genes as exemplified by TMEM176A, which showed 35-fold enrichment in the cortex and 30-fold enrichment in hippocampus of FRL animals relative to FSL.

Conclusions

These data provide significant insights into the molecular differences underlying the Flinders model, and have potential relevance to broader depression research.     

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - BSA bovine serum albumin - Bz benzoyl - EACA 6()-aminocaproic acid - HEPES N-2-hydroxyethylpiperazine-N'-propanesulfonic acid - HPLC high-performance liquid chromatography - PEG polyethylene glycol-3350 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids     

Phylogenetic analysis of the north‐east Atlantic and Mediterranean species of the genus Stenosoma (Isopoda,Valvifera, Idoteidae)

Xavier, R., Santos, A. M., Harris, D. J., Sezgin, M., Machado, M., Branco, M. (2012). Phylogenetic analysis of the north‐east Atlantic and Mediterranean species of the genus Stenosoma (Isopoda, Valvifera, Idoteidae). —Zoologica Scripta, 41, 386–399. The marine isopod genus Stenosoma is widespread in the northern hemisphere. However, 12 of its 14 known species are found within the Mediterranean basin and adjacent regions of the north‐east Atlantic and the Black Sea. Such a high level of diversity confined to a limited region of a much larger circumglobal distribution suggests that the Mediterranean region may have played a crucial role in the evolutionary history of this genus. In the present work, the phylogeny of the genus Stenosoma was investigated on the basis of DNA sequencing data from one nuclear (28SrRNA) and two mitochondrial (COI, ND4) gene fragments obtained for nine of 12 Atlantic–Mediterranean species. Divergence time estimates point to a Tethyan origin of Stenosoma and suggest that the speciation events from which stem most of the extant species took place well before the Messinian Salinity Crisis. Stenosoma spinosum and Stenosoma appendiculatum are the only exceptions, as they apparently arose within the Mediterranean during the Pleistocene. Phylogenetic reconstruction agrees with current taxonomic status of most species. However, Stenosoma capito clustered in two distinct and well‐supported clades, one composed of eastern Mediterranean and Black Sea specimens and the other by western Mediterranean and Atlantic ones. Such polyphyly suggests the existence of a previously unrecognized species, Stenosoma sp., which so far has been confounded with S. capito.     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

Effects of Beta-Alanine Supplementation on Brain Homocarnosine/Carnosine Signal and Cognitive Function: An Exploratory Study

Objectives

Two independent studies were conducted to examine the effects of 28 d of beta-alanine supplementation at 6.4 g d^-1 on brain homocarnosine/carnosine signal in omnivores and vegetarians (Study 1) and on cognitive function before and after exercise in trained cyclists (Study 2).

Methods

In Study 1, seven healthy vegetarians (3 women and 4 men) and seven age- and sex-matched omnivores undertook a brain 1H-MRS exam at baseline and after beta-alanine supplementation. In study 2, nineteen trained male cyclists completed four 20-Km cycling time trials (two pre supplementation and two post supplementation), with a battery of cognitive function tests (Stroop test, Sternberg paradigm, Rapid Visual Information Processing task) being performed before and after exercise on each occasion.

Results

In Study 1, there were no within-group effects of beta-alanine supplementation on brain homocarnosine/carnosine signal in either vegetarians (p = 0.99) or omnivores (p = 0.27); nor was there any effect when data from both groups were pooled (p = 0.19). Similarly, there was no group by time interaction for brain homocarnosine/carnosine signal (p = 0.27). In study 2, exercise improved cognitive function across all tests (P<0.05), although there was no effect (P>0.05) of beta-alanine supplementation on response times or accuracy for the Stroop test, Sternberg paradigm or RVIP task at rest or after exercise.

Conclusion

28 d of beta-alanine supplementation at 6.4g d^-1 appeared not to influence brain homocarnosine/carnosine signal in either omnivores or vegetarians; nor did it influence cognitive function before or after exercise in trained cyclists.     

Estimation of the ferrous-transferrin binding constants based on thermodynamic studies of nickel(II)-transferrin

The equilibrium constants for the binding of Ni2+ to human serum transferrin in 0.01 M hepes containing 5 mM sodium bicarbonate at 25 degrees C and pH 7.4 have been measured. The effective binding constants are log K1 = 4.10 +/- 0.15 and log K2 = 3.23 +/- 0.31 for the reactions Ni2+ + apoTr (K1) in equilibrium Ni2+-Tr. Ni2+ + Ni2+-Tr (K2) in equilibrium Ni2+-Tr-Ni2+ where the explicit terms for bicarbonate and hydrogen ion have been incorporated into the effective binding constants. Titration of both forms of mono(ferric)transferrin indicates that unlike other metal ions, Ni2+ binds preferentially to the N-terminal binding site, but that the site preference is rather small. A linear-free-energy relationship (LFER) for the complexation of Ni2+ and Fe2+ has been prepared. This LFER has been used to estimate effective binding constants of log K1 = 3.2 and log K2 = 2.5 for the ferrous-transferrin complex. These ferrous constants have been combined with the literature binding constants for ferric-transferrin to estimate formal reduction potentials of -340 mV vs. NHE for the C-terminal site and -280 mV for the N-terminal site.