Micha M.M. Wilhelmus

Transglutaminases and Transglutaminase-Catalyzed Cross-Links Colocalize with the Pathological Lesions in Alzheimer's Disease Brain

Alzheimers disease (AD) is characterized by pathological lesions, in particular senile plaques (SPs), cerebral amyloid angiopathy (CAA) and neurofibrillary tangles (NFTs), predominantly consisting of self‐aggregated proteins amyloid beta (Aβ) and tau, respectively. Transglutaminases (TGs) are inducible enzymes, capable of modifying conformational and/or structural properties of proteins by inducing molecular covalent cross‐links. Both Aβ and tau are substrates for TG cross‐linking activity, which links TGs to the aggregation process of both proteins in AD brain. The aim of this study was to investigate the association of transglutaminase 1 (TG1), transglutaminase 2 (TG2) and TG‐catalyzed cross‐links with the pathological lesions of AD using immunohistochemistry. We observed immunoreactivity for TG1, TG2 and TG‐catalyzed cross‐links in NFTs. In addition, both TG2 and TG‐catalyzed cross‐links colocalized with Aβ in SPs. Furthermore, both TG2 and TG‐catalyzed cross‐links were associated with CAA. We conclude that these TGs demonstrate cross‐linking activity in AD lesions, which suggests that both TG1 and TG2 are likely involved in the protein aggregation processes underlying the formation of SPs, CAA and/or NFTs in AD brain.

Collagen XVIII: a Novel Heparan Sulfate Proteoglycan Associated with Vascular Amyloid Depositions and Senile Plaques in Alzheimer's Disease Brains

Heparan sulfate proteoglycans (HSPGs) may play a role in the formation and persistence of senile plaques and neurofibrillary tangles in Alzheimers disease brains. Recently, it has been demonstrated that the human extracellular matrix‐associated molecule collagen XVIII is the first collagen carrying heparan sulfate side‐chains. Two variants of collagen XVIII with both different signal peptides and N‐terminal domains have been described and are referred to as the short and long form. To investigate the distribution of these variants we performed an immunohistochemical analysis by using specific well‐characterized polyclonal antibodies. Anti‐long huXVIII, a polyclonal antibody directed against the long variant of collagen XVIII, weakly stained large cortical and leptomeningeal vessels, whereas small cortical vessels remained unstained. Interestingly, all amyloid‐laden vessels and classic senile plaques were strongly stained. Anti‐all huXVIII, a polyclonal antibody directed against an epitope common to both collagen XVIII variants, intensely stained all types of cerebral blood vessels, cerebral amyloid angiopathy‐affected vessels and classic senile plaques. Collagen XVIII expression was absent in neurofibrillary tangles. We conclude that collagen XVIII is a novel heparan sulfate proteoglycan associated with vascular Aβ and classic senile plaques and that at least the long form of collagen XVIII accumulates in amyloid‐laden vessels and classic senile plaques.

Tissue transglutaminase: A novel pharmacological target in preventing toxic protein aggregation in neurodegenerative diseases

Alzheimers disease, Parkinsons disease and Huntingtons disease are neurodegenerative diseases, characterized by the accumulation and deposition of neurotoxic protein aggregates. The capacity of specific proteins to self-interact and form neurotoxic aggregates seems to be a common underlying mechanism leading to pathology in these neurodegenerative diseases. This process might be initiated and/or accelerated by proteins that interact with these aggregating proteins. The transglutaminase (TG) family of proteins are calcium-dependent enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine isopeptide bonds, which can result in both intra- and intermolecular cross-links. Intramolecular cross-links might modify self-interacting proteins, and make them more prone to aggregate. In addition, intermolecular cross-links could link self-aggregating proteins and thereby initiate and/or stimulate the aggregation process. So far, increased levels and activity of tissue transglutaminase (tTG), the best characterized member of the TG family, have been observed in many neurodegenerative diseases, and the self-interacting proteins, characteristic of Alzheimers disease, Parkinsons disease and Huntingtons disease, are known substrates of tTG. Here, we focus on the role of tTG in the initiation of the aggregation process of self-interacting proteins in these diseases, and promote the notion that tTG might be an attractive novel target for treatment of neurodegenerative diseases.

Tissue transglutaminase modulates α-synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross‐link formation between protein‐bound glutamine residues and primary amines, with Parkinsons disease‐associated α‐synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild‐type and mutant α‐synucleins using surface plasmon resonance approaches, revealing high‐affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective α‐synuclein cross‐linking, resulting predominantly in intramolecularly cross‐linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/α‐synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all α‐synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal α‐synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal β‐sheet‐containing oligomers, the tTG/α‐synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild‐type and disease mutant α‐synuclein variants. We propose that tTG cross‐linking imposes structural constraints on α‐synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross‐linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.

Presence of tissue transglutaminase in granular endoplasmic reticulum is characteristic of melanized neurons in Parkinson's disease brain.

Parkinsons disease (PD) is characterized by the accumulation of α‐synuclein aggregates and degeneration of melanized neurons. The tissue transglutaminase (tTG) enzyme catalyzes molecular protein cross‐linking. In PD brain, tTG‐induced cross‐links have been identified in α‐synuclein monomers, oligomers and α‐synuclein aggregates. However, whether tTG and α‐synuclein occur together in PD affected neurons remains to be established. Interestingly, using immunohistochemistry, we observed a granular distribution pattern of tTG, characteristic of melanized neurons in PD brain. Apart from tTG, these granules were also positive for typical endoplasmic reticulum (ER)‐resident chaperones, that is, protein disulphide isomerase, ERp57 and calreticulin, suggesting a direct link to the ER. Additionally, we observed the presence of phosphorylated pancreatic ER kinase (pPERK), a classical ER stress marker, in tTG granule positive neurons in PD brain, although no subcellular colocalization of tTG and pPERK was found. Our data therefore suggest that tTG localization to granular ER compartments is specific for stressed melanized neurons in PD brain. Moreover, as also α‐synuclein aggregates were observed in tTG granule positive neurons, these results provide a clue to the cellular site of interaction between α‐synuclein and tTG.

Antioxidative defense mechanisms controlled by Nrf2: state-of-the-art and clinical perspectives in neurodegenerative diseases

Activation of microglial cells and impaired mitochondrial function are common pathological characteristics of many neurological diseases and contribute to increased generation of reactive oxygen species (ROS). It is nowadays accepted that oxidative damage and mitochondrial dysfunction are key hallmarks of classical neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease. To counteract the detrimental effects of ROS and restore the delicate redox balance in the central nervous system (CNS), cells are equipped with an endogenous antioxidant defense mechanism consisting of several antioxidant enzymes. The production of many antioxidant enzymes is regulated at the transcriptional level by the transcription factor nuclear factor E2-related factor 2 (Nrf2). Although evidence is accumulating that activation of the Nrf2 pathway represents a promising therapeutic approach to restore the CNS redox balance by reducing ROS-mediated neuronal damage in experimental models of neurodegenerative disorders, only a few Nrf2-activating compounds have been tested in a clinical setting. We here provide a comprehensive synopsis on the role of ROS in common neurodegenerative disorders and discuss the therapeutic potential of the Nrf2 pathway.

Apolipoprotein E genotype regulates amyloid-beta cytotoxicity.

The ϵ4 allele of apolipoprotein E (ApoE) is a risk factor for Alzheimers disease (AD), whereas the ϵ2 allele may be relatively protective. Both alleles are risk factors for cerebral amyloid angiopathy (CAA)-related hemorrhages. CAA is associated with degeneration of smooth muscle cells and pericytes. Previously, we described that synthetic amyloid-β1-40 peptide (Aβ1-40) with the 22Glu→ Gln “Dutch” mutation caused pericyte death in vitro by a mechanism that involves Aβ fibril-like assembly at the cell surface. It is known that ApoE binds to Aβ and may modify its biological activities. In the present study, we evaluated the effect of ApoE on Aβ-mediated toxicity of cerebrovascular cells. We observed that cultured cells with an ϵ4/ϵ4 genotype were more vulnerable to Aβ than cultures with an ϵ3/ϵ3 or ϵ3/ϵ4 genotype. The one cell culture with the ϵ2/ϵ3 genotype was relatively resistant to Aβ compared with other cultures. Furthermore, we observed a dose-dependent protective effect of native ApoE against Aβ-mediated toxicity of cerebrovascular cells and, in addition, ApoE ϵ2/ϵ3 cells secreted more ApoE protein compared with cells with other ApoE genotypes, in particular, compared with ϵ4/ϵ4 cells. Thus, the disparity between ApoE genotype and Aβ-mediated toxicity might be related to differences in the cellular capacity to secrete ApoE. The present data suggest that one mechanism by which ApoE may alter the risk for AD is a genotype-dependent regulation of Aβ cytotoxicity, possibly via variations in its secretion levels, whereby extracellular ApoE may bind to Aβ and thereby modify Aβ-mediated cell death.

Involvement and interplay of Parkin, PINK1, and DJ1 in neurodegenerative and neuroinflammatory disorders.

The involvement of parkin, PINK1, and DJ1 in mitochondrial dysfunction, oxidative injury, and impaired functioning of the ubiquitin-proteasome system (UPS) has been intensively investigated in light of Parkinsons disease (PD) pathogenesis. However, these pathological mechanisms are not restricted to PD, but are common denominators of various neurodegenerative and neuroinflammatory disorders. It is therefore conceivable that parkin, PINK1, and DJ1 are also linked to the pathogenesis of other neurological diseases, including Alzheimers disease (AD) and multiple sclerosis (MS). The importance of these proteins in mechanisms underlying neurodegeneration is reflected by the neuroprotective properties of parkin, DJ1, and PINK1 in counteracting oxidative stress and improvement of mitochondrial and UPS functioning. This review provides a concise overview on the cellular functions of the E3 ubiquitin ligase parkin, the mitochondrial kinase PINK1, and the cytoprotective protein DJ1 and their involvement and interplay in processes underlying neurodegeneration in common neurological disorders.

Parkinson's disease-associated parkin colocalizes with Alzheimer's disease and multiple sclerosis brain lesions

Parkin is implicated in the pathogenesis of Parkinsons disease. Furthermore, parkin targets misfolded proteins for degradation and protects cells against various forms of cellular stress, including unfolded-protein and oxidative stress. This points towards a protective role of parkin in neurological disorders in which these stressors are implicated, including Alzheimers disease (AD) and multiple sclerosis (MS). Here, we assessed parkin distribution in AD and MS brain tissue using immunohistochemistry. In AD brains, parkin colocalized with classic senile plaques and amyloid-laden vessels as well as astrocytes associated with both lesions. Similarly, we observed enhanced astrocytic parkin immunoreactivity in MS lesions, particularly in inflammatory lesions. Furthermore, parkin mRNA expression was increased in an astrocytoma cell line after free radical exposure. Our data indicate that parkin is upregulated in AD and MS brain tissue and might represent a defense mechanism to counteract stress-induced damage in AD and MS pathogenesis.

Association of Parkinson disease-related protein PINK1 with Alzheimer disease and multiple sclerosis brain lesions

Mitochondrial dysfunction and oxidative stress are hallmarks of various neurological disorders, including multiple sclerosis (MS), Alzheimer disease (AD), and Parkinson disease (PD). Mutations in PINK1, a mitochondrial kinase, have been linked to the occurrence of early onset parkinsonism. Currently, various studies support the notion of a neuroprotective role for PINK1, as it protects cells from stress-mediated mitochondrial dysfunction, oxidative stress, and apoptosis. Because information about the distribution pattern of PINK1 in neurological diseases other than PD is scarce, we here investigated PINK1 expression in well-characterized brain samples derived from MS and AD individuals using immunohistochemistry. In control gray matter PINK1 immunoreactivity was observed in neurons, particularly neurons in layers IV-VI. Astrocytes were the most prominent cell type decorated by anti-PINK1 antibody in the white matter. In addition, PINK1 staining was observed in the cerebrovasculature. In AD, PINK1 was found to colocalize with classic senile plaques and vascular amyloid depositions, as well as reactive astrocytes associated with the characteristic AD lesions. Interestingly, PINK1 was absent from neurofibrillary tangles. In active demyelinating MS lesions we observed a marked astrocytic PINK1 immunostaining, whereas astrocytes in chronic lesions were weakly stained. Taken together, we observed PINK1 immunostaining in both AD and MS lesions, predominantly in reactive astrocytes associated with these lesions, suggesting that the increase in astrocytic PINK1 protein might be an intrinsic protective mechanism to limit cellular injury.