Irène M. Riederer

The role of the ubiquitin proteasome system in Alzheimer's disease

Today, Alzheimers disease (AD) is one of the most important age-related neurodegenerative diseases, but its etiology remains still unknown. Since the discovery that the hallmark structures of this disease i.e. the formation of amyloid fibers could be the product of ubiquitin-mediated protein degradation defects, it has become clear that the ubiquitin–proteasome system (UPS), usually essential for protein repair, turnover and degradation, is perturbed in this disease. Different aspects of normal and pathological aging are discussed with respect to protein repair and degradation via the UPS, as well as consequences of a deficit in the UPS in AD. Selective protein oxidation may cause protein damage, or protein mutations may induce a dysfunction of the proteasome. Such events eventually lead to activation of cell death pathways and to an aberrant aggregation or incorporation of ubiquitinated proteins into hallmark structures. Aggresome formation is also observed in other neurodegenerative diseases, suggesting that an activation of similar mechanisms must occur in neurodegeneration as a basic phenomenon. It is essential to discuss therapeutic ways to investigate the UPS dysfunction in the human brain and to identify specific targets to hold or stop cell decay.

Differential Changes in Synaptic Proteins in the Alzheimer Frontal Cortex with Marked Increase in PSD-95 Postsynaptic Protein

We investigated how synaptic plasticity is related to the neurodegeneration process in the human dorsolateral prefrontal cortex. Pre- and postsynaptic proteins of Brodmanns area 9 from patients with Alzheimers disease (AD) and age-matched controls were quantified by immunohistochemical methods and Western blots. The main finding was a significant increase in the expression of postsynaptic density protein PSD-95 in AD brains, revealed on both sections and immunoblots, while the expression of spinophilin, associated to spines, remained quantitatively unchanged despite qualitative changes with age and disease. Presynaptic protein alpha-synuclein indicated an increased immunohistochemical level, while synaptophysin remained unchanged. MAP2, a somatodendritic microtubule protein, as well as AD markers such as amyloid-beta protein and phosphorylated protein tau showed an increased expression on immunosections in AD. Altogether these changes suggest neuritic and synaptic reorganization in the process of AD. In particular, the significant increase in PSD-95 expression suggests a change in NMDA receptors trafficking and may represent a novel marker of functional significance for the disease.

Ubiquitination and cysteine nitrosylation during aging and Alzheimer's disease

Protein oxidation and ubiquitination of brain proteins are part of mechanisms that modulate protein function or that inactivate proteins and target misfolded proteins to degradation. In this study, we focused on brain aging and on mechanism involved in neurodegeneration such as events occurring in Alzheimers disease (AD). The goal was to identify differences in nitrosylated proteins - at cysteine residues, and in the composition of ubiquinated proteins between aging and Alzheimers samples by using a proteomic approach. A polyclonal anti-S-nitrosyl-cysteine, a mono- and a polyclonal anti-ubiquitin antibody were used for the detection of modified or ubiquitinated proteins in middle-aged and aged human entorhinal autopsy brains tissues of 14 subjects without neurological signs and 8 Alzheimers patients. Proteins were separated by one- and two-dimensional gel electrophoresis and analyzed by Coomassie blue and immuno-blot staining. We identified that the glial fibrillary acidic and tau proteins are more ubiquitinated in brain tissues of Alzheimers patients. Furthermore, glial fibrillary proteins were also found in nitrosylated state and further characterized by 2D Western blots and identified. Since reactive astrocytes localized prominently around senile plaques one can speculate that elements of plaques such as beta-amyloid proteins may activate surrounding glial elements and proteins.

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications.

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimers disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimers disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.

Skin fibroblast model to study an impaired glutathione synthesis: Consequences of a genetic polymorphism on the proteome

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Increased postsynaptic density protein-95 expression in the frontal cortex of aged cognitively impaired rats

In the present work we studied synaptic protein concentrations in relation to behavioral performance. Long-Evans rats, aged 22-23 months, were classified for individual expression of place memory in the Morris water maze, in reference to young adults. Two main subgroups of aged rats were established: the Aged cognitively Unimpaired (AU) had search accuracy within the range (percent of time in training sector within mean+2 SEM) of young rats and the Aged cognitively Impaired (AI) rats had search accuracy below this range. Samples from the hippocampus and frontal cortex of all the AI, AU and young rats were analyzed for the expression of postsynaptic protein PSD-95 by Image J analysis of immunohistochemical data and by Western blots. PSD-95 expression was unchanged in the hippocampus, but, together with synaptophysin, was significantly increased in the frontal cortex of the AI rats. A significant correlation between individual accuracy (time spent in the training zone) and PSD-95 expression was observed in the aged group. No significant effect of age or PSD-95 expression was observed in the learning of a new position. All together, these data suggest that increased expression of PSD-95 in the frontal cortex of aged rats co-occurs with cognitive impairment that might be linked to functional alterations extending over frontal networks.

Selective changes in the neurofilament and microtubule cytoskeleton of NF-H/LacZ mice

This study focused mainly on changes in the microtubule cytoskeleton in a transgenic mouse where β‐galactosidase fused to a truncated neurofilament subunit led to a decrease in neurofilament triplet protein expression and a loss in neurofilament assembly and abolished transport into neuronal processes in spinal cord and brain. Although all neurofilament subunits accumulated in neuronal cell bodies, our data suggest an increased solubility of all three subunits, rather than increased precipitation, and point to a perturbed filament assembly. In addition, reduced neurofilament phosphorylation may favor an increased filament degradation. The function of microtubules seemed largely unaffected, in that tubulin and microtubule‐associated proteins (MAP) expression and their distribution were largely unchanged in transgenic animals. MAP1A was the only MAP with a reduced signal in spinal cord tissue, and differences in immunostaining in various brain regions corroborate a relationship between MAP1A and neurofilaments.

Phosphorylation-dependent dimerization and subcellular localization of islet-brain 1/c-Jun N-terminal kinase-interacting protein 1.

Islet‐brain 1 [IB1; also termed c‐Jun N‐terminal kinase (JNK)‐interacting protein 1 (JIP‐1] is involved in the apoptotic signaling cascade of JNK and functions as a scaffold protein. It organizes several MAP kinases and the microtubule‐transport motor protein kinesin and relates to other signal‐transducing molecules such as the amyloid precursor protein. Here we have identified IB1/JIP‐1 using different antibodies that reacted with either a monomeric or a dimeric form of IB1/JIP‐1. By immunoelectron microscopy, differences in the subcellular localization were observed. The monomeric form was found in the cytoplasmic compartment and is associated with the cytoskeleton and with membranes, whereas the dimeric form was found in addition in nuclei. After treatment of mouse brain homogenates with alkaline phosphatase, the dimeric form disappeared and the monomeric form decreased its molecular weight, suggesting that an IB1/JIP‐1 dimerization is phosphorylation dependent and that IB1 exists in several phospho‐ forms. N‐methyl‐D‐aspartate receptor activation induced a dephosphorylation of IB1/JIP‐1 in primary cultures of cortical neurons and reduced homodimerization. In conclusion, these data suggest that IB1/JIP‐1 monomers and dimers may differ in compartmental localization and thus function as a scaffold protein of the JNK signaling cascade in the cytoplasm or as a transcription factor in nuclei.

Differential subcellular localization of phosphorylated neurofilament and tau proteins in degenerating neurons of the human entorhinal cortex.

&NA; A panel of novel monoclonal antibodies was tested on the human entorhinal cortex for the recognition of age‐ and disease‐related changes of neurofilament proteins (NF). Several antibodies identified phosphorylated NF‐H subunit, which occurred preferentially in those aged between 60 and 80 years and were localized in degenerating neurons. Such neurons also contained neurofibrillary tangles, but neurofilament aggregates did not co‐localize with tangles, nor did the quantity nor the number of NF‐positive neurons correlate with the severity of Alzheimers disease. This points to a susceptibility of NF in a subset of neurons for phosphor‐ylation‐ and metabolically related morphological changes during neurodegeneration.

P4-275: Postsynaptic density protein PSD-95 increases in Alzheimer entorhinal and frontal cortex as a marker of synaptic changes

is a postsynaptic scaffolding protein that is responsible for the clustering of glycine and subsets of GABAA receptors with the microtubule cytoskeleton at the inhibitory synapse. Methods: To quantify markers of excitatory and inhibitory synapses in AD compared with controls in pathologically affected and spared areas of the brain. To discern differences in the levels of these markers under increasing pathological severity. Results: N-cadherin and -catenin levels were markedly higher (P 0.01) in AD cases (n 15) than in controls (n 15), whereas gephyrin was significantly reduced (P 0.01). AD cases were scored according to pathological severity of AD by accounting for neuronal loss, tangle and plaque load, and gliosis. A score of 0 indicated no pathology; 1, mild or modest pathology; 2, moderate pathology; and 3, severe pathology, for each area studied. N-cadherin and -catenin showed higher levels with mild severity but gephyrin levels declined at a moderate index of pathological severity. Conclusions: These results might suggest not only an imbalance of excitatory and inhibitory synapses in AD, but a specific synaptic dysfunction, preferentially affecting excitatory synapses followed by inhibitory synapses. This synaptic dysfunction may contribute to the regional neurodegeneration in AD.