Dariusz Koziorowski

Ferritin as an important player in neurodegeneration.

Oxidative stress is considered one of the pathways leading to neuronal death in neurodegenerative disease. Many published studies aimed to assess the possible role of iron in this process but no consensus has been reached. On the other hand little is known about the role played by the main iron storage protein - ferritin. In this review we discuss the data obtained using several methods - Mössbauer spectroscopy, electron microscopy and ELISA - from human brain tissue both in controls and in four neurodegenerative disorders - Parkinsons (PD) and Alzheimers disease, progressive supranuclear palsy and neuroferritinopathy. Iron may only cause oxidative stress injury when it is available as labile iron for Fenton reaction. This may be related to the decreased ability of ferritin to retain iron within the iron core of ferritin. This happens in PD and in neuroferritinopathy. In PD there is a decrease in the concentration of L ferritin, while in neuroferritinopathy there is a genetically induced mutation in L ferritin causing its loss of function. We discuss the importance of the ratio H/L ferritin and its changes in neurodegeneration.

Iron as a cause of Parkinson disease - a myth or a well established hypothesis?

Iron is considered to be a possible trigger of oxidative stress leading to neurodegeneration. This mechanism of neuronal death is proposed as a cause of Parkinson disease. Although most of researchers agree with this, controversies remain regarding the amounts of iron needed for this process. According to non destructive methods of assessment of the concentration of the total iron in substantia nigra, there is no difference between PD and control. However there is no need for an increase of the total iron in parkinsonian SN to trigger the oxidative stress but only of the non-ferritin bound labile iron. Our recent studies suggest an increase of this iron in PD SN. This finding corresponds well to a decrease of L-ferritin concentration in parkinsonian SN and also to a difference of the size of iron core of ferritin between PD and control SN. The significance of these finding will be discussed.

Heterozygous PINK1 p.G411S increases risk of Parkinson’s disease via a dominant-negative mechanism

See Gandhi and Plun-Favreau (doi:10.1093/aww320) for a scientific commentary on this article. It has been postulated that heterozygous mutations in recessive Parkinson’s genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson’s disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson’s disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson’s disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype.

Novel A18T and pA29S substitutions in α-synuclein may be associated with sporadic Parkinson's disease.

OBJECTIVE Mutations in the α-synuclein-encoding gene SNCA are considered as a rare cause of Parkinsons disease (PD). Our objective was to examine the frequency of the SNCA point mutations among PD patients of Polish origin. METHODS Detection of the known SNCA point mutations A30P (c.88G>C), E46K (c.136G>A) and A53T (c.157A>T) was performed either using the Sequenom MassArray iPLEX platform or by direct sequencing of the SNCA exons 2 and 3. As the two novel substitutions A18T (c.52G>A) and A29S (c.85G>T) were identified, their frequency in a control population of Polish origin was assessed and in silico analysis performed to investigate the potential impact on protein structure and function. RESULTS We did not observe the previously reported point mutations in the SNCA gene in our 629 PD patients; however, two novel potentially pathogenic substitutions A18T and A29S were identified. Each variant was observed in a single patient presenting with a typical late-onset sporadic PD phenotype. Although neither variant was observed in control subjects and in silico protein analysis predicts a damaging effect for A18T and pA29S substitutions, the lack of family history brings into question the true pathogenicity of these rare variants. CONCLUSIONS Larger population based studies are needed to determine the pathogenicity of the A18T and A29S substitutions. Our findings highlight the possible role of rare variants contributing to disease risk and may support further screening of the SNCA gene in sporadic PD patients from different populations.

Subthalamic deep brain stimulation for the treatment of Parkinson disease

BACKGROUND AND PURPOSE The role of subthalamic nucleus deep brain stimulation (STN DBS) in the treatment of Parkinson disease (PD) is well established. The authors present a group of patients diagnosed with PD who were treated with STN DBS. MATERIAL AND METHODS Between 2008 and 2009, 32 female and 34 male patients with PD were treated with STN DBS. Mean age at implantation was 57 ± 12 years. PD lasted from 6 to 21 years (mean 10 years). Patients were qualified for the surgery according to the CAPSIT-PD criteria. The STN was identified with direct and indirect methods. Macrostimulation and microrecording for STN identification were used in all cases. A unilateral STN DBS system was implanted in two cases and bilateral implantation was performed among rest of the group. Outcome was assessed six months after implantation. Results : The mean reduction of UPDRS III score among 51 patients who underwent follow-up was 45% (5-89%). Reduction of levodopa consumption varied from 15 to 100%. Infection forced the authors to remove the DBS system in one case four months after implantation. Skin erosion above the internal pulse generator was noted in four cases. CONCLUSIONS Cardinal symptoms of Parkinsons disease can be safely and effectively treated with STN DBS in selected group of patients.

Levodopa “drug holiday” with amantadine infusions as a treatment of complications in Parkinson's disease

The loss of beneficial effect of levodopa due to progression of the disease and alteration of receptor sensitivity makes the treatment of the advanced stadium of Parkinsons disease (PD) very difficult. In the past “drug holidays” was used in attempt to resensitize dopamine receptors in the striatum to make the treatment easier. However possible serious complications like neuroleptical malignant‐like syndrome discouraged the use of this procedure. Intravenous administration of amantadine, another antiparkinsonian medication during “drug holidays,” procedure could be a solution for this problem. We studied 12 patients with PD suffering from complication of the therapy. Daily dose of Levodopa used as monotherapy before amantadine infusions ranged between 700 and 2,000 mg. Levodopa was discontinued for 3 days and during that time amantadine sulfate intravenous was administrated. After “drug holidays” levodopa in the same dose as before treatment was resumed. An assessment of the parkinsonian condition was performed with Unified Parkinsons Disease Rating Scale before “drug holidays” 2 days after and 1, 2, 3, 4, 5, 6 months later. The follow‐up study demonstrated a significant improvement both in the motor condition and complication of therapy. The improvement after therapy was maintained up to 4 month. The levodopa “drug holidays” with amantadine infusion is a valuable option in the therapy of advanced stages of PD.

Iron and ferritin in hippocampal cortex and substantia nigra in human brain — Implications for the possible role of iron in dementia

The concentrations of iron and of ferritin, the main iron-binding compound in the brain, as well as the sizes of the iron cores of ferritin were assessed in hippocampal cortex (Hip) and substantia nigra (SN) from human control brains, using Mössbauer spectroscopy (MS), ELISA and electron microscopy. 8 Hip and 20 SN samples were measured by MS, 11 Hip and 11 SN were used for ELISA, and the size of the iron cores of ferritin was assessed from measurements of 50 iron cores from Hip-ferritin and 50 iron cores from SN-ferritin. The average concentration of iron in Hip was found to be about one third of that in SN, as was the concentration of H-ferritin, yet L-ferritin was less than one fifth in Hip compared to SN. The size of the average iron core in Hip was assessed to be about 3.1 nm and about 3.7 nm in SN. These results may point to different iron metabolism in these areas, suggesting faster iron turnover in Hip.

Multimodal Learning and Intelligent Prediction of Symptom Development in Individual Parkinson's Patients.

We still do not know how the brain and its computations are affected by nerve cell deaths and their compensatory learning processes, as these develop in neurodegenerative diseases (ND). Compensatory learning processes are ND symptoms usually observed at a point when the disease has already affected large parts of the brain. We can register symptoms of ND such as motor and/or mental disorders (dementias) and even provide symptomatic relief, though the structural effects of these are in most cases not yet understood. It is very important to obtain early diagnosis, which can provide several years in which we can monitor and partly compensate for the disease’s symptoms, with the help of various therapies. In the case of Parkinson’s disease (PD), in addition to classical neurological tests, measurements of eye movements are diagnostic. We have performed measurements of latency, amplitude, and duration in reflexive saccades (RS) of PD patients. We have compared the results of our measurement-based diagnoses with standard neurological ones. The purpose of our work was to classify how condition attributes predict the neurologist’s diagnosis. For n = 10 patients, the patient age and parameters based on RS gave a global accuracy in predictions of neurological symptoms in individual patients of about 80%. Further, by adding three attributes partly related to patient ‘well-being’ scores, our prediction accuracies increased to 90%. Our predictive algorithms use rough set theory, which we have compared with other classifiers such as Naïve Bayes, Decision Trees/Tables, and Random Forests (implemented in KNIME/WEKA). We have demonstrated that RS are powerful biomarkers for assessment of symptom progression in PD.

Data Mining and Machine Learning on the Basis from Reflexive Eye Movements Can Predict Symptom Development in Individual Parkinson’s Patients

We are still not in a position to understand most of the brain’s deeper computational properties. As a consequence, we also do not know how brain processes are affected by nerve cell deaths in neurodegenerative diseases (ND). We can register symptoms of ND such as motor and/or mental disorders (dementias) and even provide symptomatic relief, though the structural effects of these are in most cases not yet understood. Fortunately, with early diagnosis there are often many years of disease progression with symptoms that, when they are precisely monitored, may result in improved therapies. In the case of Parkinson’s disease, measurements of eye movements can be diagnostic. In order to better understand their relationship to the underlying disease process, we have performed measurements of reflexive eye movements in Parkinson’s disease (PD) patients. We have compared our measurements and algorithmic diagnoses with experts’ diagnoses. The purpose of our work was to find universal rules, using rough set theory, to classify how condition attributes predict the neurologist’s diagnosis. Prediction of individual UPDRS values only from reflexive saccade (RS) latencies was not possible. But for n = 10 patients, the patient’s age, latency, amplitude, and duration of RS gave a global accuracy in individual patients’ UPRDS predictions of about 80%, based on cross-validation. This demonstrates that broadening the spectrum of physical measurements and applying data mining and machine learning (ML) can lead to a powerful biomarker for symptom progression in Parkinson’s.

The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity

BackgroundMutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson’s disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future.MethodsHere, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients’ fibroblasts and in cell-based, biochemical assays.ResultsUnder endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity.ConclusionsHere we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients’ fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development.