Mitochondrial impairment observed in fibroblasts from South African Parkinson's disease patients with parkin mutations.

Parkinson's disease (PD), defined as a neurodegenerative disorder, is characterized by the loss of dopaminergic neurons in the substantia nigra in the midbrain. Loss-of-function mutations in the parkin gene are a major cause of autosomal recessive, early-onset PD. Parkin has been implicated in the maintenance of healthy mitochondria, although previous studies show conflicting findings regarding mitochondrial abnormalities in fibroblasts from patients harboring parkin-null mutations. The aim of the present study was to determine whether South African PD patients with parkin mutations exhibit evidence for mitochondrial dysfunction. Fibroblasts were cultured from skin biopsies obtained from three patients with homozygous parkin-null mutations, two heterozygous mutation carriers and two wild-type controls. Muscle biopsies were obtained from two of the patients. The muscle fibers showed subtle abnormalities such as slightly swollen mitochondria in focal areas of the fibers and some folding of the sarcolemma. Although no differences in the degree of mitochondrial network branching were found in the fibroblasts, ultrastructural abnormalities were observed including the presence of electron-dense vacuoles. Moreover, decreased ATP levels which are consistent with mitochondrial dysfunction were observed in the patients' fibroblasts compared to controls. Remarkably, these defects did not manifest in one patient, which may be due to possible compensatory mechanisms. These results suggest that parkin-null patients exhibit features of mitochondrial dysfunction. Involvement of mitochondria as a key role player in PD pathogenesis will have important implications for the design of new and more effective therapies.

Interaction of acetylcholinesterase with the G4 domain of the laminin alpha1-chain.

Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neurite outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChE's PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val(2718)-Arg-Lys-Arg-Leu(2722), Tyr(2738)-Tyr(2739), Tyr(2789)-Ile-Lys-Arg-Lys(2793) and Val(2817)-Glu-Arg-Lys(2820), on the mouse laminin alpha1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps extensively with laminin's heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg(90)-Glu-Leu-Ser-Glu-Asp(95) on the omega loop, and also the involvement of Pro(40)-Pro-Val(42), Arg(46) (linked to Glu(94) by a salt bridge) and the hexapeptide Asp(61)-Ala-Thr-Thr-Phe-Gln(66). Epitope analysis, using CLiPS technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro(40)-Pro-Met-Gly-Pro-Arg-Arg-Phe(48) (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro(78)-Gly-Phe-Glu-Gly-Thr-Glu(84) and Pro(88)-Asn-Arg-Glu-Leu-Ser-Glu-Asp(95). Antibodies that recognized other features of the PAS area but not the Arg(90)-Gly-Leu-Ser-Glu-Asp(95) motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse.

New Insights Into Autophagy Dysfunction Related to Amyloid Beta Toxicity and Neuropathology in Alzheimer's Disease.

The fine control of neuronal proteostasis is an essential element that preserves cell viability. Advancing age is a major risk factor for Alzheimer's disease (AD), and autophagy is thought to dictate normal and pathological aging through intricate molecular machinery controlling protein aggregation. Although the role of autophagy dysfunction in AD is known, the dynamic changes during the progression of the disease remain unclear. Recent studies have provided new insight into the molecular mechanisms that link defective autophagy and cellular fate, underscoring the pathogenic events associated with AD. Here, we will focus on recent studies that underpin a distinct role for autophagy deficits and highly localized autophagic defects, impacting primarily the amyloidogenic pathway activity. By uniquely assessing the dynamic changes in key proteins during the disease progression in the context of the autophagy machinery function and amyloid beta toxicity, specifically, a connect between protein degradation failure and cell death susceptibility is revealed which may suggest new avenues for the development of better targeted therapeutic interventions.

Autophagy and the invisible line between life and death.

For a considerable time cell death has been considered to represent mutually exclusive states with cell death modalities that are governed by their inherent and unique mode of action involving specific molecular entities and have therefore been studied primarily in isolation. It is now, however, becoming increasingly clear that these modalities are regulated by similar pathways and share a number of initiator and effector molecules that control both cell death as well as cell survival mechanisms, demanding a newly aligned and integrative approach of cell death assessment. Frequently cell death is triggered through a dual action that incorporates signaling events associated with more than one death modality. Apoptosis and necrosis regularly co-operate in a tightly balanced interplay that involves autophagy to serve context dependently either as a pro-survival or a pro-death mechanism. In this review we will assess current cell death modalities and their molecular overlap with the goal of clarifying the controversial role of autophagy in the cell death response. By dissecting the key molecular pathways and their positioning within a network of regulatory signalling hubs and checkpoints we discuss a distinct approach that integrates autophagy with a resultant cell death manifestation. In doing so, former classifications of cell death modalities fade and reveal the intricate molecular proportions and complexities of the cell death response that may contribute towards an enhanced means of cell death control.

Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients.

Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.

Neurodegenerative disorders: dysregulation of a carefully maintained balance?

The aggregation of misfolded proteins has long been regarded as a pathological event in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease. However, the exact molecular mechanisms that govern protein metabolism that may lead to toxicity remain largely unclear. Originally targeted as the causative agent, it has since become evident that aggregation formation may not be necessary for disease progression and studies show that they may even serve functional and protective roles. Although the focus has since shifted to the toxicity of intermediate protein species preceding aggregation formation, many questions remain: Is the blame for the neural destruction to be put on one event alone, or rather on a state of cellular disequilibrium resulting from multiple events? If the cause is multifactorial, then what triggers the toxic cascade and how can this be targeted therapeutically? In order to understand the origin of toxicity, the exact underlying mechanism and impact of each contributing process must be assessed. Therefore, the structural properties, mechanism of formation, cytotoxic and/or protective effects, as well as the clinical impact of protein intermediates and aggregates will be reviewed here with the goal to establish a neurodegenerative disease model aimed at improving current therapeutics, which may ultimately contribute towards improved treatment modalities.

Non-enzymatic developmental functions of acetylcholinesterase--the question of redundancy.

Despite in vitro demonstrations of non-enzymatic morphogenetic functions in acetylcholinesterase (AChE), the AChE knockout phenotype is milder than might be expected, casting doubt upon the relevance of such functions in vivo. Functional redundancy is a possible explanation. Using in vitro findings that AChE is able to bind to laminin-111, together with detailed information about the interaction sites, as well as an epitope analysis of adhesion-inhibiting anti-AChE mAbs, we have used molecular docking and bioinformatics techniques to explore this idea, investigating structurally similar molecules that have a comparable spatiotemporal expression pattern in the embryonic nervous system. On this basis, molecules with which AChE could be redundant are the syndecans, glypicans, perlecan, the receptor tyrosine kinase Mer, and the low-density lipoprotein receptor. It is also highly likely that AChE may be redundant with the homologous neuroligins, although there is no evidence that the latter are expressed before synaptogenesis. AChE was observed to dock with Gas6, the ligand for Mer, as well as with apolipoprotein E3 (but not apolipoprotein E4), both at the same site as the laminin interaction. These findings suggest that AChE may show direct functional redundancy with one or more of these molecules; it is also possible that it may itself have a unique function in the stabilization of the basement membrane. As basement membrane molecules are characterized by multiple molecular interactions, each contributing cumulatively to the construction and stability of the network, this may account for AChE's apparently promiscuous interactions, and also for the survival of the knockout.