Myelin-associated proteins are involved in the formation and stabilization of myelin sheaths. In addition, they prevent axon regeneration and plasticity in the adult brain. Recent evidence suggests that the expression of certain myelin-associated proteins (e.g. Nogo-A) can be regulated by synaptic activity or by over-expression after neural lesions in brain syndromes such as temporal lobe epilepsy. However, no studies on Alzheimer disease (AD) have been reported in which cell loss and significant synaptic reorganization occurs. In the present study, we analyze in detail the expression of Nogo-A in the hippocampal formation in normal human aging and in AD. Our results indicate that Nogo-A is expressed by oligodendrocytes and neurons in the aged hippocampal formation. In addition, both granule cells and mossy fiber connections are also labeled in the old-aged hippocampi. Interestingly, Nogo-A is over-expressed by hippocampal neurons in AD and is associated with beta-amyloid deposits in senile plaques. Taken together, our results reinforce the hypothesis that Reticulon proteins such as Nogo-A participate in the neuronal responses stemming from hippocampal formation during senescence, and particularly in AD. These findings also indicate that Reticulon proteins could be considered as new putative drug targets in therapies of neurodegenerative disorders.

The conversion of cellular prion protein (PrP c )

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.

Prnp knockout mice that overexpress an amino-truncated form of PrPc (deltaPrP) are ataxic and display cerebellar cell loss and premature death. Studies on the molecular and intracellular events that trigger cell death in these mutants may contribute to elucidate the functions of PrPc and to the design of treatments for prion disease. Here we examined the effects of Bcl-2 overexpression in neurons on the development of the neurological syndrome and cerebellar pathology of deltaPrP. We show that deltaPrP overexpression activates the stress-associated kinases ERK1-2 in reactive astroglia, p38 and the phosphorylation of p53, which leads to the death of cerebellar neurons in mutant mice. We found that the expression of deltaPrP in cell lines expressing very low levels of PrPc strongly induces the activation of apoptotic pathways, thereby leading to caspase-3 activation and cell death, which can be prevented by coexpressing Bcl-2. Finally, we corroborate in vivo that neuronal-directed Bcl-2 overexpression in deltaPrP mice (deltaPrP Bcl-2) markedly reduces caspase-3 activation, glial activation, and neuronal cell death in cerebellum by improving locomotor deficits and life expectancy.

The physiological functions of PrPC remain enigmatic, but the central domain, comprising highly conserved regions of the protein may play an important role. Indeed, a large number of studies indicate that synthetic peptides containing residues 106–126 (CR) located in the central domain (CD, 95–133) of PrPC are neurotoxic. The central domain comprises two chemically distinct subdomains, the charge cluster (CC, 95–110) and a hydrophobic region (HR, 112–133). The aim of the present study was to establish the individual cytotoxicity of CC, HR and CD. Our results show that only the CD peptide is neurotoxic. Biochemical, Transmission Electron Microscopy and Atomic Force Microscopy experiments demonstrated that the CD peptide is able to activate caspase-3 and disrupt the cell membrane, leading to cell death.

The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.

Membrane transporters significantly modulate membrane permeability of endobiotics and xenobiotics, such as bile acids and drugs, respectively. Various in vitro methods have been established for both ATP-binding cassette (ABC) transporters to examine cellular efflux and uptake, and for solute carriers (SLC) to examine cellular uptake of substrates. Cell-based systems are the models of choice to test drug-transporter interactions as well as drug-drug interactions for research and regulatory purposes, albeit, for low passive permeability substrates of ABC transporters, vesicular uptake assays are also recommended. Commercially available pre-plated cells (e.g., immortalized or transfected) offer a useful alternative to in-house cell culture. Three main methods are known to manufacture pre-plated cultures: regular culture medium with vacuum seal, cryopreserved delivery, and the solid shipping media technology. The regular culture medium and the solid shipping media technologies provide ready-to-use models for end users. Models expressing a broad selection of transporters are available in pre-plated formats for absorption, distribution, metabolism, excretion, and toxicity (ADMETox) studies. Conversely, the application and utility of pre-plated cultures coupled with personal experiences have not been extensively covered in published research papers or reviews, despite availability and significant use of pre-plated products in the pharmaceutical industry. In this overview, we will briefly describe: 1) in vitro tools commonly used for ADMETox testing; 2) methods employed in manufacturing, shipment and preparation of pre-plated cell lines; 3) cell-membrane barrier models currently available in pre-plated format to reproduce passage restriction of physiological barriers to certain compounds; and 4) recommended pre-plated cell lines overexpressing uptake transporters for ADMETox applications.