Ubiquitylome profiling of Parkin-null brain reveals dysregulation of calcium homeostasis factors ATP1A2, Hippocalcin and GNA11, reflected by altered firing of noradrenergic neurons.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear how selective neural circuits become vulnerable and finally undergo atrophy. We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein targets of Parkin activity, an ubiquitin E3 ligase. Thus, we now used aged Parkin-knockout (KO) mouse brain for a global quantification of ubiquitylated peptides by mass spectrometry (MS). This approach confirmed the most abundant substrate to be VDAC3, a mitochondrial outer membrane porin that modulates calcium flux, while uncovering also >3-fold dysregulations for neuron-specific factors. Ubiquitylation decreases were prominent for Hippocalcin (HPCA), Calmodulin (CALM1/CALML3), Pyruvate Kinase (PKM2), sodium/potassium-transporting ATPases (ATP1A1/2/3/4), the Rab27A-GTPase activating protein alpha (TBC1D10A) and an ubiquitin ligase adapter (DDB1), while strong increases occurred for calcium transporter ATP2C1 and G-protein subunits G(i)/G(o)/G(Tr). Quantitative immunoblots validated elevated abundance for the electrogenic pump ATP1A2, for HPCA as neuron-specific calcium sensor, which stimulates guanylate cyclases and modifies axonal slow afterhyperpolarization (sAHP), and for the calcium-sensing G-protein GNA11. We assessed if compensatory molecular regulations become insufficient over time, leading to functional deficits. Patch clamp experiments in acute Parkin-KO brain slices indeed revealed alterations of the electrophysiological properties in aged noradrenergic locus coeruleus (LC) neurons. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in sAHP and shortening of action potential duration, without modulation of KCNQ potassium currents. These findings indicate altered calcium-dependent excitability in a PARK2 model of PD, mediated by diminished turnover of potential Parkin targets such as ATP1A2 and HPCA. The data also identified further novel Parkin substrate candidates like SIRT2, OTUD7B and CUL5. Our elucidation of neuron-specific mechanisms of PD pathogenesis helps to explain the known exceptional susceptibility of noradrenergic and dopaminergic projections to alterations of calcium homeostasis and its mitochondrial buffering.

Imbalances in protein homeostasis caused by mutant desmin.

AIMS: We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. METHODS: We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy. RESULTS: We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB-crystallin and Hsp27. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies. CONCLUSIONS: Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.

Sequence variations in the NDUFA1 gene encoding a subunit of complex I of the respiratory chain.

NDUFA1 is one of the 36 nuclear genes encoding subunits of the mitochondrial complex I involved in the respiratory chain. The human NDUFA1 has been cloned, completely sequenced and mapped to Xq24. In the present study, we searched for sequence variations in NDUFA1 as causative defects in complex I deficiency using genomic DNA of 152 patients with various clinical phenotypes. The patient sample consisted of 54 patients (46 male and 8 female) with Leber heriditary optic neuropathy (LHON) from 48 unrelated families from Germany and 98 patients (72 male and 26 female) with biochemically proven complex I deficiency including Leigh syndrome. Patient DNA was used to amplify all three exons, including the exon/intron boundaries and the promoter region of NDUFA1 for heteroduplex analysis and direct sequencing. In the 152 patients tested, no mutation was found that could be related to any of the disease phenotypes included. However, three single-nucleotide polymorphisms (SNPs) located in the promoter region (SNP G/C at nt -71 and SNP T/C at nt -189) and in intron 1 (SNP T/G nt 1454) were discovered. Allele frequencies of the SNPs were estimated in a German and Estonian control population and compared to complex I-deficient patients. There was no significant difference between the control population, the LHON patients, or the severely affected patients with complex I deficiency, excluding an association of the polymorphisms with the diseases. Our results suggest that mutations in NDUFA1 do not cause the gender difference observed in clinically severe and complex phenotypes with complex I deficiency.

Serological evidence of Chlamydia pneumoniae lipopolysaccharide antibodies in atherosclerosis of various vascular regions.

BACKGROUND: The role of Chlamydia pneumoniae in the pathogenesis of atherosclerosis has so far mainly been investigated in patients suffering from coronary heart disease; the other vascular regions have virtually been ignored. The aim of this study was to carry out a statistical survey of serological markers of a C. pneumoniae infection in patients with different patterns of atherosclerosis manifestation. PATIENTS AND METHODS: 340 patients were examined for the atherosclerotic alteration of peripheral arteries of the lower limbs, carotid arteries and coronary arteries by ultrasound scan and/or angiography. Immunoglobulin(Ig)G and IgA-rELISA were used to measure chlamydial lipopolysaccharide antibodies. Species determination was performed using the IgG micro-immunofluorescence test. RESULTS: 24.0% of atherosclerotic cases (A) and 52.3% of controls (C) were negative for C. pneumoniae lipopolysaccharide antibodies (p = 0.00002). By contrast, 45.1% of atherosclerotic cases and 16.9% of controls were positive for both IgG and IgA (p = 0.00002). The mean antibody titers of the atherosclerosis group were higher than in the control group (IgG positive xAIgG = 344, xCIgG = 272; IgG and IgA positive xAIgG = 576, xCIgG = 486 and xAIgA = 120, xCIgA = 91). Concerning atherosclerosis manifestation in various vascular regions, no significant differences were found between IgG and IgA antibody titers and prevalence. CONCLUSIONS: The results show that a persistent C, pneumoniae infection with evidence of lipopolysaccharide immunoglobulin G and A is equally associated with the atherosclerotic alteration of coronary arteries, carotid arteries and peripheral arterial occlusive disease, irrespective of the severity of atherosclerosis and with no predisposition to any particular vascular region.

Genomic organization of the human complex I 13-kDa subunit gene NDUFA5.

We have characterized the human gene NDUFA5 encoding a 13-kDa subunit of the mitochondrial respiratory chain complex I (NADH: ubiquinone oxidoreductase). The gene contains 5 exons and 4 introns, and spans 14 kb of genomic DNA. In the untranscribed region we observed potential transcription factor binding sites. We determined a single nucleotide variant (C/T) at -318, and its frequency in the German population. The functional gene was localised by FISH to 7q31 and by radiation hybrid panel near marker D7S648 in YAC 883_a_2.

[Grönblad-Strandberg syndrome from the angiological viewpoint]. / Das Grönblad-Strandberg-Syndrom aus angiologischer Sicht.

HISTORY AND CLINICAL FINDINGS: A 42-year-old man was admitted for treatment of peripheral vascular disease in the left leg (stage III of Fontaine). A year before he had undergone a right aortofemoral bypass operation. On admission there was stenosis of the left pelvic axis and bilateral femoral artery occlusion. In addition there were changes in the skin with abnormal folds, loss of elasticity and yellowish spots over the sides of the neck and the flexor surfaces of all large joints. In addition vision in the left eye was impaired. These findings suggested connective tissue disease involving the skin, eye and arterial system. INVESTIGATIONS: Routine haematological tests were normal as were clotting parameters. Serum concentration of GOT, GPT, gamma-GT were slightly increased. There was a dysproteinaemia with raised HDL and LDL levels. Resting electrocardiogram was normal, showing sinus rhythm and left axis deviation. The crurobrachial pressure index was clearly abnormal: 0.6 on the right and 0.5 on the left. Angiography of the pelvic and left arteries revealed long-segment femoral and partial lower-leg occlusions bilaterally. Abdominal sonography indicated diffuse parenchymal calcifications in both kidneys and angioid streaks on bilateral fundoscopy. Skin biopsy showed defects of elastic fibres and perivascular inflammatory infiltration, while capillary microscopy revealed twisting of the capillaries, most of them with normal lumen. These findings taken together indicated pseudoxanthoma elasticum (PXE) or Grönblad-Strandberg syndrome. TREATMENT AND COURSE: A thrombendarterectomy was performed on the left superficial femoral artery, after which the left popliteal artery became palpable, the pressure indices for the left leg were slightly better, and the patient was discharged home without further complications and improved leg perfusion. CONCLUSION: Possible cardiovascular involvement had to be taken into account in patients with PXE, and long-term angiological monitoring is indicated.

[Coronary vessel anomalies in adults--harmless variation or changes with clinical significance]. / Koronaranomalien bei Erwachsenen--harmlose Variation oder Veränderungen mit Krankheitswert.

A review of 1097 consecutive coronary angiograms performed for evaluation of angina pectoris or valvular dysfunctions showed 3 patients with arteriovenous fistulas (0.25%), 6 patients with malpositions (0.55%), 29 patients with aneurysms (2.65%) and 59 patients with myocardial bridging (5.4%). The clinical significance of coronary artery anomalies as variation or malformation was evaluated. A malformation has been found in Bland-White-Garland syndrome, arteriovenous fistulas, congenital coronary stenosis and atresia. In case of clinical symptoms surgical correction should be considered. There is a controversy on the clinical significance of muscular bridging. Further clinical investigations are needed for a better understanding of coronary artery anomalies.

[Static and dynamic infrared thermometry and thermography in malignant melanoma of the uvea and conjunctiva]. / Statische und dynamische Infrarotthermometrie und -thermographie beim malignen Melanom der Uvea und Konjunktiva.

BACKGROUND: Thermometry and -graphy prove asymmetries of the circulation. Contact-free thermometry of the cornea is applied to find out whether this method can contribute to differential diagnosis of ocular melanomas. MATERIALS AND METHODS: Under standardized conditions the temperature of the cornea was measured in 30 patients with malignant melanoma of the choroid and the conjunctiva and 35 healthy subjects. The instruments were a handpyrometer (HPM, Messgerätewerk, Magdeburg) and the thermovision camera of AGA Infrared System, Sweden, with dynamic recording in colour. RESULTS: In healthy subjects there are no significant asymmetries in temperature under standardized conditions. The temperature of the cornea is elevated in both malignant melanomas of the choroid and the conjunctiva. CONCLUSIONS: Thermometry and -graphy of the cornea can contribute to the differentiation of malignant ocular melanomas from other ophthalmological diseases. They are also suited in long term observation of the course of the melanomas.