Luisa Gregori

Ubiquitin, cellular inclusions and their role in neurodegeneration

Covalent binding of ubiquitin to proteins marks them for degradation by the ubiquitin/ATP-dependent pathway. This pathway plays a major role in the breakdown of abnormal proteins that result from oxidative stress, neurotoxicity and mutations. Failure to eliminate ubiquitinated proteins disrupts cellular homeostasis, causing degeneration. Inclusions containing ubiquitinated proteins are commonly detected in many neurological disorders. These aggregates are mostly cytosolic; nevertheless, ubiquitinated inclusions are found in endosomes/lysosomes in Alzheimers disease and prion encephalopathies, and in nuclei in disorders associated with CAG/polyglutamine repeats, such as Huntingtons disease and spinocerebellar ataxias. Ubiquitinated aggregates must result from a malfunction or overload of the ubiquitin/ATP-dependent pathway or from structural changes in the protein substrates, halting their degradation. Prevention of protein aggregation in these diseases might offer new therapeutic leads.

Mutant ubiquitin expressed in Alzheimer’s disease causes neuronal death1

Ubiquitin‐B+1 (UBB+1) is a mutant ubiquitin that accumulates in the neurones of patients with Alzheimers disease (AD). Here we report on the biochemical and functional differences between ubiquitin and UBB+1 and the effect of the mutant protein on neuronal cells. UBB+1 lacks the capacity to ubiquiti‐nate, and although it is ubiquitinated itself, UBB+1 is not degraded by the ubiquitin‐proteasomal system and is quite stable in neuronal cells. Overexpression of UBB+1 in neuroblastoma cells significantly induces nuclear fragmentation and cell death. Our results demonstrate that accumulation of UBB+1 in neurones is detrimental and may contribute to neuronal dysfunction in AD patients.—de Vrij, F. M. S., Sluijs, J. A., Gregori, L., Fischer, D. F., Hermens, W. T. J. M. C., Goldgaber, D., Verhaagen, J., van Leeuwen, F. W., Hol, E. M. Mutant ubiquitin expressed in Alzheimers disease causes neuronal death. FASEB J. 15, 2680–2688 (2001)

Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer’s disease pathology ☆

Nucleolin is a major multifunctional nuclear phosphoprotein that is phosphorylated by Cdc2 kinase in mitosis and that participates in a number of cellular processes. The monoclonal antibody TG-3 generated against neurofibrillary tangles (NFT) found in Alzheimers disease (AD) is highly specific for mitotic cells in culture. We here demonstrate that phosphorylation of nucleolin by Cdc2 kinase generates the TG-3 epitope. The unique pool of TG-3 immunoreactive nucleolin appears abruptly during the prophase. It is associated with chromosomes through the metaphase and it gradually disappears during separation of chromosomes and exit from mitosis. In the brain, nucleolin was localized not only to nuclei but also to neuronal cytoplasm, and it is a marker for early NFT. In patients with AD, Cdc2 phosphorylated nucleolin was present in NFT. These findings suggest that phosphorylation of nucleolin by Cdc2 kinase is a critical event and the point of convergence of two distinct pathways, mitosis and neurodegeneration.

Sequestration of Amyloid β‐Peptidea

Amyloid β‐protein, or β/A4, is a 4‐kilodalton peptide that forms poorly soluble extracellular depositions of amyloid in brains and leptomeninges of patients with Alzheimers disease (AD), Downs syndrome (DS), and hereditary cerebral hemorrhage with amyloidosis‐Dutch type (HCHWA‐D). β/A4 peptide is a derivative of a large transmembrane glycoprotein (APP) and is found in the extracellular space, i.e., in the cerebrospinal fluid and serum of individuals with and without AD and in the conditioned media of many different cells grown in culture.1 The mechanism by which normally produced amyloid β peptide forms extracellular aggregates in patients is unknown. One possible explanation is a failure of a mechanism for removal of the β/A4 peptide that prevents this highly aggregating peptide from forming extracellular amyloid depositions.

Selection of peptides binding to the amyloid b-protein reveals potential inhibitors of amyloid formation

Alzheimers disease (AD) is a neurodegenerative disorder characterized by extracellular amyloid plaques, cerebrovascular amyloid deposits, intracellular neurofibrillary tangles, and neuronal loss. Amyloid deposits are composed of insoluble fibers of a 39–43 amino acid peptide named the amyloid β-protein (Aβ). Neuropathological and genetic studies provide strong evidence of a key role for Aβ amyloidosis in the pathogenesis of AD. Therefore, an obvious pharmacological target for treatment of AD is the inhibition of amyloid growth and/or inhibition of amyloid function. We took an unbiased approach to generate new inhibitors of amyloid formation by screening a FliTrx™ combinatorial peptide library for Aβ binding peptides and identified four groups of peptides with different Aβ binding motifs. In addition, we designed and examined peptides mimicking the Aβ binding domain of transthyretin (TTR). Our results showed that Aβ binding peptides selected from FliTrx™ peptide library and from TTR-peptide analogs are capable of inhibiting Aβ aggregation and Aβ deposition in vitro. These properties demonstrate that binding of selected peptides to the amyloid β-protein may provide potent therapeutic compounds for the treatment AD.

A processing pathway for amyloid β-protein precursor generates a secreted form with an intact carboxyl terminus

Extracellular deposition of amyloid β-protein (Aβt) is a key neuropathological feature of Alzheimers disease (AD), Downs syndrome and hereditary cerebral hemorrhage with amyloidoses-Dutch type (HCHWA-D). Aβis part of a larger transmembrane glycoprotein, the amyloid Pprotein precursor (AβPP). Two processing pathways are known for AβPP. In the secretory pathway AβPP is secreted following cleavage within Aβ thereby preventing Aβ formation. The endosomal/lysosomal pathway generates intracellular carboxyl terminal fragments that contain the Aβ region. Recently, Aβ has been detected in human cerebrospinal fluid, plasma and in conditioned medium from a variety of cell culture systems. This indicates that Aβ is formed by a normal processing pathway that has not yet been understood. To investigate this pathway we have expressed AβPP751 and two disease related point mutants of AβPP resulting in glu693 -to-gln andval717 -to-ile in the baculovirus expression system. In each case we detected secreted AβPP molecules ...