Application of an Image Cytometry Protocol for Cellular and Mitochondrial Phenotyping on Fibroblasts from Patients with Inherited Disorders.

Cellular phenotyping of human dermal fibroblasts (HDFs) from patients with inherited diseases provides invaluable information for diagnosis, disease aetiology, prognosis and assessing of treatment options. Here we present a cell phenotyping protocol using image cytometry that combines measurements of crucial cellular and mitochondrial parameters: (1) cell number and viability, (2) thiol redox status (TRS), (3) mitochondrial membrane potential (MMP) and (4) mitochondrial superoxide levels (MSLs). With our protocol, cell viability, TRS and MMP can be measured in one small cell sample and MSL on a parallel one. We analysed HDFs from healthy individuals after treatment with various concentrations of hydrogen peroxide (H2O2) for different intervals, to mimic the physiological effects of oxidative stress. Our results show that cell number, viability, TRS and MMP decreased, while MSL increased both in a time- and concentration-dependent manner. To assess the use of our protocol for analysis of HDFs from patients with inherited diseases, we analysed HDFs from two patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD), one with a severe clinical phenotype and one with a mild one. HDFs from both patients displayed increased MSL without H2O2 treatment. Treatment with H2O2 revealed significant differences in MMP and MSL between HDFs from the mild and the severe patient. Our results establish the capacity of our protocol for fast analysis of cellular and mitochondrial parameters by image cytometry in HDFs from patients with inherited metabolic diseases.

The Hsp60 folding machinery is crucial for manganese superoxide dismutase folding and function.

Even though the deleterious effects of increased reactive oxygen species (ROS) levels have been implicated in a variety of neurodegenerative disorders, the triggering events that lead to the increased ROS and successive damages are still ill-defined. Mitochondria are the key organelles controlling the ROS balance, being their main source and also counteracting them by the action of the ROS scavenging system. Mitochondria, moreover, control the presence of ROS-damaged proteins by action of the protein quality control (PQC) system. One of its components is the mitochondrial chaperone Hsp60 assisting the folding of a subset of mitochondrial matrix proteins. Mutations in Hsp60 cause a late onset form of the neurodegenerative disease hereditary spastic paraplegia (SPG13). In this study, we aimed to address the molecular consequences of Hsp60 shortage. We here demonstrate that a heterozygous knockout Hsp60 model that recapitulates features of the human disease and exhibits increased oxidative stress in neuronal tissues. Moreover, we indicate that the increase of ROS is, at least in part, due to impaired folding of the manganese superoxide dismutase (MnSOD), a key antioxidant enzyme. We observed that the Hsp60 and MnSOD proteins interact. Based on these results, we propose that MnSOD is a substrate of the Hsp60 folding machinery and that under conditions of diminished availability of Hsp60, MnSOD is impaired in reaching the native state. This suggests a possible link between Hsp60-dependent PQC and the ROS scavenging systems that may have the function to increase ROS production under conditions of folding stress.

Protein expression studies of desmoplakin mutations in cardiomyopathy patients reveal different molecular disease mechanisms.

Mutations in the gene for desmoplakin (DSP) may cause arrhythmogenic right ventricular cardiomyopathy (ARVC) and Carvajal syndrome (CS). Desmoplakin is part of all desmosomes, which are abundantly expressed in both myocardial and epidermal tissue and serve as intercellular mechanical junctions. This study aimed to investigate protein expression in myocardial and epidermal tissue of ARVC and CS patients carrying DSP mutations in order to elucidate potential molecular disease mechanisms. Genetic investigations identified three ARVC patients carrying different heterozygous DSP mutations in addition to a homozygous DSP mutation in a CS patient. The protein expression of DSP in mutation carriers was evaluated in biopsies from myocardial and epidermal tissue by immunohistochemistry. Keratinocyte cultures were established from skin biopsies of mutation carriers and characterized by reverse transcriptase polymerase chain reaction, western blotting, and protein mass spectrometry. The results showed that the mutation carriers had abnormal DSP expression in both myocardial and epidermal tissue. The investigations revealed that the disease mechanisms varied accordingly to the specific types of DSP mutation identified and included haploinsufficiency, dominant-negative effects, or a combination hereof. Furthermore, the results suggest that the keratinocytes cultured from patients are a valuable and easily accessible resource to elucidate the effects of desmosomal gene mutations in humans.

Decreased expression of the mitochondrial matrix proteases Lon and ClpP in cells from a patient with hereditary spastic paraplegia (SPG13).

The mitochondrial chaperonin heat shock protein 60 (Hsp60) assists the folding of a subset of proteins localized in mitochondria and is an essential component of the mitochondrial protein quality control system. Mutations in the HSPD1 gene that encodes Hsp60 have been identified in patients with an autosomal dominant form of hereditary spastic paraplegia (SPG13), a late-onset neurodegenerative disorder characterized by a progressive paraparesis of the lower limbs. The disease-associated Hsp60-(p.Val98Ile) protein, encoded by the c.292G>A HSPD1 allele, has reduced chaperonin activity, but how its expression affects mitochondrial functions has not been investigated. We have studied mitochondrial function and expression of genes encoding mitochondrial chaperones and proteases in a human lymphoblastoid cell line and fibroblast cells from a patient who is heterozygous for the c.292G>A HSPD1 allele. We found that both the c.292G>A RNA transcript and the corresponding Hsp60-(p.Val98Ile) protein were present at comparable levels to their wild-type counterparts in SPG13 patient cells. Compared with control cells, we found no significant cellular or mitochondrial dysfunctions in SPG13 patient cells by assessing the mitochondrial membrane potential, cell viability, and sensitivity toward oxidative stress. However, a decreased expression of the mitochondrial protein quality control proteases Lon and ClpP, both at the RNA and protein level, was demonstrated in SPG13 patient cells. We propose that decreased levels of mitochondrial proteases Lon and ClpP may allow Hsp60 substrate proteins to go through more folding attempts instead of being prematurely degraded, thereby supporting productive folding in cells with reduced Hsp60 chaperonin activity. In conclusion, our studies with SPG13 patient cells expressing the functionally impaired mutant Hsp60 chaperonin suggest that reduction of the degradative activity of the protein quality control system may represent a previously unrecognized cellular adaptation to reduced chaperone function.

Influence of culture pH on survival of Lactobacillus reuteri subjected to freeze-drying.

L. reuteri was cultivated at pH 5 and 6. The cells were harvested at 0.5, 2.5 and 4.5 h after entering the stationary phase and their viability after freeze-drying was compared with their viability prior to freeze-drying. The highest viability--approximately 80% of the viability prior to freeze-drying--was obtained for cells from the pH 5 cultures harvested 2.5 h after entering the stationary phase. The time after entering the stationary phase did not influence the viability of the cells from the pH 6 cultures where the viability was approximately 50% irrespective of harvest time. Product formation was the same for pH 5 and pH 6 grown cells, whereas the pH 6 grown cells exhibited a more elongated morphology.