Abnormal expression of synaptic proteins and neurotrophin-3 in the Down syndrome mouse model Ts65Dn.

Down syndrome (DS) results from triplication of the whole or distal part of human chromosome 21. Persons with DS suffer from deficits in learning and memory and cognitive functions in general, and, starting from early development, their brains show dendritic and spine structural alterations and cell loss. These defects concern many cortical brain regions as well as the hippocampus, which is known to play a critical role in memory and cognition. Most of these abnormalities are reproduced in the mouse model Ts65Dn, which is partially trisomic for the mouse chromosome 16 that is homologous to a portion of human chromosome 21. Thus, Ts65Dn is widely utilized as an animal model of DS. To better understand the molecular defects underlying the cognitive and particularly the memory impairments of DS, we investigated whether the expression of several molecules known to play critical roles in long-term synaptic plasticity and long-term memory in a variety of species is dysregulated in either the neonatal brain or adult hippocampus of Ts65Dn mice. We found abnormal expression of the synaptic proteins synaptophysin, microtubule-associated protein 2 (MAP2) and cyclin-dependent kinase 5 (CDK5) and of the neurotrophin-3 (NT-3). Both the neonatal brain and adult hippocampus revealed significant abnormalities. These results suggest that a dysregulation in the expression of neurotrophins as well as proteins involved in synaptic development and plasticity may play a potential role in the neural pathology of DS in humans.

Mechanisms of memory stabilization and de-stabilization.

Memories become stabilized through a time-dependent process that requires gene expression and is commonly known as consolidation. During this time, memories are labile and can be disrupted by a number of interfering events, including electroconvulsive shock, trauma and other learning or the transient effect of drugs such as protein synthesis inhibitors. Once consolidated, memories are insensitive to these disruptions. However, they can again become fragile if recalled or reactivated. Reactivation creates another time-dependent process, known as reconsolidation, during which the memory is restabilized. Here we discuss some of the questions currently debated in the field of memory consolidation and reconsolidation, the molecular and anatomical requirements for both processes and, finally, their functional relationship.

The consolidation of new but not reactivated memory requires hippocampal C/EBPbeta.

Long-term memory formation consists of multiple phases. A new memory is initially labile and sensitive to disruption by a variety of interfering events or agents. To become stable, this new memory undergoes a process known as consolidation, which, in the case of declarative memories, occurs within the medial temporal lobes and requires gene expression. When recalled, memories re-enter a new phase of vulnerability and seem to require a reconsolidation process in order to be maintained. Here we show that consolidation but not reconsolidation of inhibitory avoidance memory requires the expression of the transcription factor CCAAT enhancer binding protein beta (C/EBPbeta) in the hippocampus. Furthermore, in the same region, de novo protein synthesis is not essential for memory reconsolidation. C/EBPbeta is an evolutionarily conserved genetic marker that has a selective role in the consolidation of new but not reactivated memories in the hippocampus.

Fornix-dependent induction of hippocampal CCAAT enhancer-binding protein [beta] and [delta] Co-localizes with phosphorylated cAMP response element-binding protein and accompanies long-term memory consolidation.

The cAMP response element-binding protein (CREB) is an evolutionarily conserved transcription regulator essential for long-term memory formation. It is not known, however, whether the molecular events downstream of CREB activation are also conserved. An early, cAMP-dependent event necessary for learning-related long-term synaptic plasticity in the invertebrate Aplysia californica is the induction of the transcription factor CCAAT enhancer-binding protein (C/EBP). Here we show that two homologs in the rat, C/EBPbeta and C/EBPdelta, are induced at discrete times after inhibitory avoidance learning and co-localize with phosphorylated CREB in the hippocampus. This induction is blocked by fornix lesions, which are known to disrupt activation of CREB in the hippocampus and to impair memory consolidation. These results indicate that C/EBPs are evolutionarily conserved components of the CREB-dependent gene cascade activated in long-term memory.

Genes to remember.

It has been known for several decades that the formation of long-term memory requires gene expression. In recent years, the use of genetic and molecular approaches has led to the identification and characterization of genes and molecules that play a fundamental role in the biological mechanisms underlying learning and memory. From these studies, it appears that molecules and molecular mechanisms essential for the process of memory have been conserved throughout evolution. The cyclic AMP (cAMP)-dependent activation pathway and a cAMP-dependent cascade of gene expression have been shown to be essential for memory formation in Aplysia californica, Drosophila melanogaster and rodents. Moreover, members of the transcription factor family cAMP response element binding proteins (CREBs) seem to represent key molecules for transforming incoming information into long-term memory. Here, we review the studies showing that conserved molecules and biological mechanisms are engaged in simple and complex forms of memory.

C/EBP is an immediate-early gene required for the consolidation of long-term facilitation in Aplysia.

The consolidation of long-term memory requires protein and mRNA synthesis. A similar requirement has been demonstrated for learning-related synaptic plasticity in the gill-withdrawal reflex of Aplysia. The monosynaptic component of this reflex can be reconstituted in vitro, where it undergoes both short- and long-term increases in synaptic strength in response to serotonin (5-HT), a neurotransmitter released during behavioral sensitization, a simple form of learning. As with sensitization, the long-term synaptic modification is characterized by a brief consolidation period during which gene expression is required. We find that during this phase, the transcription factor Aplysia CCAAT enhancer-binding protein (ApC/EBP) is induced rapidly by 5-HT and by cAMP, even in the presence of protein synthesis inhibitors. Blocking the function of ApC/EBP blocks long-term facilitation selectively without affecting the short-term process. These data indicate that cAMP-inducible immediate-early genes have an essential role in the consolidation of stable long-term synaptic plasticity in Aplysia.

Quality control of ER synthesized proteins: an exposed thiol group as a three-way switch mediating assembly, retention and degradation.

Plasma cells secrete IgM only in the polymeric form: the C-terminal cysteine of the mu heavy chain (Cys575) is responsible for both intracellular retention and assembly of IgM subunits. Polymerization is not quantitative, and part of IgM is degraded intracellularly. Neither chloroquine nor brefeldin A (BFA) inhibits degradation, suggesting that this process occurs in a pre-Golgi compartment. Degradation of IgM assembly intermediates requires Cys575: the monomeric IgMala575 mutant is stable also when endoplasmic reticulum (ER) to Golgi transport is blocked by BFA. Addition of the 20 C-terminal residues of mu to the lysosomal protease cathepsin D is sufficient to induce pre-Golgi retention and degradation of the chimeric protein: the small amounts of molecules which exit from the ER are mostly covalent dimers. By contrast, when retained by the KDEL sequence, cathepsin D is stable in the ER, indicating that retention is not sufficient to cause degradation. Replacing the C-terminal cysteine with serine restores transport through the Golgi. As all chimeric cathepsin D constructs display comparable protease activity in vitro, their different fates are not determined by gross alterations in folding. Thus, also out of its normal context, the mu chain Cys575 plays a crucial role in quality control, mediating assembly, retention and degradation.

Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents.

There are several demonstrations that misfolded or unassembled proteins are not transported along the secretory pathway, but are retained intracellularly, generally in the endoplasmic reticulum. For instance, B lymphocytes synthesize but do not secrete IgM, and only the polymeric form of IgM is secreted by plasma cells. The C-terminal cysteine of the mu heavy chain of secreted IgM (residue 575) is involved in the intracellular retention of unpolymerized IgM subunits. Here we report that the addition of reducing agents to the culture medium, at concentrations which do not affect cell viability, terminal glycosylation, or retention of proteins in the endoplasmic reticulum through the KDEL mechanism, induces secretion of IgM assembly intermediates by both B and plasma cells. Free joining (J) chains, which are not normally secreted by plasma cells unless as part of IgM or IgA, are also secreted in the presence of reducing agents. We propose a role for free thiol groups in preventing the unhindered transport of proteins through the secretory pathway. Under the scheme, assembly intermediates interact through their thiol groups between themselves and/or with unknown proteins of the endoplasmic reticulum. Such interactions may be prevented by altering the intracellular redox potential or by site-directed mutagenesis of the relevant cysteine residue(s).

Lymphocyte subpopulations in the neonate: high percentage of ANAE+ cells with low avidity for sheep erythrocytes.

In the present study we have stained for alfa-naphthyl acetate esterase (ANAE) cord blood lymphocytes (CBL) as well as cord blood E rosetting cells. The results obtained showed that the percentage of E rosettes (E+) is lower in cord blood than in adult peripheral blood when rosetting is carried out with untreated sheep red blood cells (SRBC), while there is no significant difference if SRBC are previously treated with 2-aminoethylisothiouronium bromide (AET). ANAE-positive cells (A+) were higher in cord than in adult blood. ANAE staining of E+ cells showed that CBL include a high percentage of A+ cells with low avidity for SRBC which could represent immature lymphocytes related to the T-cell lineage.

Lymphocyte subpopulations in the neonate: a subset of HNK-1-, OKT3-, OKT8+ lymphocytes displays natural killer activity.

It has been recently reported that cord blood lymphocytes (CBL) contain a subpopulation of OKT8+, sheep erythrocyte-rosetting negative (E-) cells not detectable in adult peripheral blood lymphocytes (a-PBL). The present studies were undertaken to characterize this subset of lymphocytes functionally and phenotypically. OKT8+ cells were purified from E-depleted CBL by negative selection on nylon-wool columns as well as by positive selection on plates coated with rabbit antibody to murine IgG (panning). The purified CBL displayed natural killer (NK) activity against K562 erythroleukemic cells. Although most of these CBL were large granular lymphocytes, they lacked typical NK markers such as HNK-1 and OKM1 surface antigens. Most OKT8+, OKT3- cells were also OKT10+, Ia+ and had the receptor for peanut agglutinin. These CBL may represent a stage along the differentiation pathway leading to mature NK or T cells.