Characterization and Molecular Profiling of PSEN1 Familial Alzheimer's Disease iPSC-Derived Neural Progenitors

Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer''s disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.     

Differential Protein Expression in Aortic Smooth Muscle Cells Cultured from Newborn and Aged Rats

Atherosclerosis is a complex disease in which smooth muscle cells (SMC) play a fundamental role. Work from several laboratories has suggested that in experimental models of atheromatosis SMC heterogeneity is important in the establishment of intimal thickening. Moreover, it has been shown that SMC cultured from different situations in vivo maintain distinct phenotypic features in vitro. In order to find proteins differentially expressed in SMC cultured from newborn and aged rats, total protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), high-resolution maps were built, and differentially expressed spots were identified by automatic computer analysis. Of the 14 differentially expressed protein spots, 4 were present in SMC of newborn and 10 in SMC of old animals; we describe their molecular weights and isoelectric points. One of these proteins (expressed only in cultured SMC of old rats) was successfully microsequenced for 16 amino acids and it was found identical to cellular retinol-binding protein. This result provides, to our knowledge, the first suggestion that retinoids are implicated in the differentiation and aging of vascular SMC.     

Beta‐amyloid 1‐42 monomers,but not oligomers,produce PHF‐like conformation of Tau protein

The mechanistic relationship between amyloid β1‐42 (Aβ1‐42) and the alteration of Tau protein are debated. We investigated the effect of Aβ1‐42 monomers and oligomers on Tau, using mice expressing wild‐type human Tau that do not spontaneously develop Tau pathology. After intraventricular injection of Aβ1‐42, mice were sacrificed after 3 h or 4 days. The short‐lasting treatment with Aβ monomers, but not oligomers, showed a conformational PHF‐like change of Tau, together with hyperphosphorylation. The same treatment induced increase in concentration of GSK3 and MAP kinases. The inhibition of the kinases rescued the Tau changes. Aβ monomers increased the levels of total Tau, through the inhibition of proteasomal degradation. Aβ oligomers reproduced all the aforementioned alterations only after 4 days of treatment. It is known that Aβ1‐42 monomers foster synaptic activity. Our results suggest that Aβ monomers physiologically favor Tau activity and dendritic sprouting, whereas their excess causes Tau pathology. Moreover, our study indicates that anti‐Aβ therapies should be targeted to Aβ1‐42 monomers too.     

Importance of introns in the growth regulation of mRNA levels of the proliferating cell nuclear antigen gene.

The steady-state mRNA levels of the proliferating cell nuclear antigen (PCNA) gene are growth regulated. We have begun to identify the elements in the human PCNA gene that participate in its growth regulation by transfecting appropriate constructs in BALB/c3T3 cells. The results can be summarized as follows. (i) The 400 base pairs of the 5'-flanking sequence of the human PCNA gene upstream of the preferred cap site are sufficient for directing expression of a heterologous cDNA (S. Travali, D.-H. Ku, M. G. Rizzo, L. Ottavio, R. Baserga, and B. Calabretta, J. Biol. Chem. 264:7466-7472, 1989). (ii) Intron 4 is necessary for the proper regulation of PCNA mRNA levels in G0 cells. Removal of intron 4 leads to abnormally high levels of PCNA mRNA in serum-deprived cells, although the shortened PCNA gene with its own promoter is still responsive to serum stimulation. (iii) The presence of introns also increases the steady-state levels of PCNA mRNA in proliferating cells. These results are especially interesting for two reasons: (i) because of the extensive sequence similarities among introns and between introns and exons of the human PCNA gene, and (ii) because, usually, the presence of introns leads to increased expression, whereas in this case, removal of intron 4 caused an increase in mRNA levels, and this occurred only in quiescent cells.     

Phenotypic plasticity in sun orientation of sandhoppers

Summary Littoral sandhoppers perform zonal orientation mainly by means of sun orientation, which in Mediterranean populations was shown to be inborn, associated with a genetically determined directional tendency adapted to the shoreline of each population. In case of changes of the shoreline the genetic heterogeneity of populations and learning ability may cooperate in providing adaptation both at the population and at the individual level. This hypothesis has been tested carrying on studies on the ontogenesis of the reactionnorm of this behavioural trait inTalitrus saltator (Montagu) (Crustacea: Amphipoda) of a Tyrrhenian population which from previous results shows a well adapted seaward orientation. Sun orientation was analysed away from the sea shore, both in laboratory-raised individuals of different ages and in individuals captured in the field, by repeated releases on dry flat sand or in a glass bowl. Results showed variability within and between individuals of directional tendency, which varies in the individual life depending on age and experience. Particularly, age seems to bring canalization as variability within individuals tends to decrease with age. In order to evaluate the role of experience, we trained individuals to direct themselves towards a direction different from that genetically determined. The majority of the individuals showed a learning ability in the training situation. The importance of phenotypic plasticity in the oriented behaviour of sandhoppers is discussed.Dedicated in gratitude to Prof. Dr. Drs. h.c. M. Lindauer     

Sagittal Diameter Minus Subcutaneous Thickness. An Easy-to-Obtain Parameter That Improves Visceral Fat Prediction

ARMELLINI FABIO, MAURO ZAMBONI, TAMARA HARRIS, ROCCO MICCIOLO, OTTAVIO BOSELLO. Sagittal diameter minus subcutaneous thickness. An easy-to-obtain parameter that improves visceral fat prediction. Two groups of 99 and 98 women were studied to test if correcting sagittal diameter by subtracting the thickness of subcutaneous abdominal adipose tissue improves its degree of association with visceral adipose tissue. The first group (age, 40 ± 14 years; body mass index [BMI], 36 ± 6 kg/m²) was used to calculate the predictive equations for visceral adipose tissue. The second group (age, 43 ± 14 years; BMI, 37 ± 6 kg/m²) was used for cross-validation. Various anthropometric parameters were measured by ultrasound and computed tomography. Correlation coefficients with single-slice visceral adipose tissue area, after sagittal diameter was corrected by subtracting subcutaneous thickness, rose from 0.63 to 0.72 in the first group and from 0.64 to 0.71 in the second group. The standard error of residuals of the regression formula for visceral adipose tissue area was 10% lower with modified sagittal diameter than with sagittal diameter alone. During cross-validation, the standard error of differences was 5% lower with modified sagittal diameter. The visceral adipose tissue estimate was also less biased by the size of the area when sagittal diameter minus subcutaneous thickness was used. Results show that subtracting the thickness of abdominal subcutaneous adipose tissue from sagittal diameter significantly improves the predictive power of sagittal diameter for visceral adipose tissue and could be a useful tool for epidemiological studies.     

Presynaptic CaMKII is necessary for synaptic plasticity in cultured hippocampal neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional enzyme that is very critical for synaptic plasticity and memory formation. Although significant progress has been made in understanding the role of postsynaptic CaMKII in synaptic plasticity, very little is known about its presynaptic function during plasticity changes. Here we report that KN-93, a membrane-permeable CaMKII inhibitor, blocked glutamate-induced increases in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and the number of presynaptic functional boutons in cultured hippocampal pyramidal neurons. In addition, presynaptic injection of the membrane-impermeable CaMKII inhibitor peptide 281-309 blocked synaptic plasticity induced by tetanus, glutamate, or NO/cGMP pathway activation as expressed by long-lasting increases in EPSC amplitude and functional presynaptic boutons. Presynaptic injection of CaMKII itself coupled with weak tetanus produced an immediate and long-lasting enhancement of EPSC amplitude. Thus, the present results conclusively prove that presynaptic CaMKII is essential for synaptic plasticity in cultured hippocampal neurons.     

Synergism between curdlan and GM-CSF confers a strong inflammatory signature to dendritic cells

A simultaneous engagement of different pathogen recognition receptors provides a tailor-made adaptive immunity for an efficient defense against distinct pathogens. For example, cross-talk of TLR and C-type lectin signaling effectively shapes distinct gene expression patterns by integrating the signals at the level of NF-κB. In this study, we extend this principle to a strong synergism between the dectin-1 agonist curdlan and an inflammatory growth factor, GM-CSF. Both together act in synergy in inducing a strong inflammatory signature that converts immature dendritic cells (DCs) to potent effector DCs. A variety of cytokines (IL-1β, IL-6, TNF-α, IL-2, and IL-12p70), costimulatory molecules (CD80, CD86, CD40, and CD70), chemokines (CXCL1, CXCL2, CXCL3, CCL12, CCL17), as well as receptors and molecules involved in fugal recognition and immunity such as Mincle, dectin-1, dectin-2, and pentraxin 3 are strongly upregulated in DC treated simultaneously with curdlan and GM-CSF. The synergistic effect of both stimuli resulted in strong IκBα phosphorylation, its rapid degradation, and enhanced nuclear translocation of all NF-κB subunits. We further identified MAPK ERK as one possible integration site of both signals, because its phosphorylation was clearly augmented when curdlan was coapplied with GM-CSF. Our data demonstrate that the immunomodulatory activity of curdlan requires an additional signal provided by GM-CSF to successfully initiate a robust β-glucan-specific cytokine and chemokine response. The integration of both signals clearly prime and tailor a more effective innate and adaptive response against invading microbes and fungi.