Sabrina Giraudo

Residual substantia nigra neuromelanin in Parkinson’s disease is cross-linked to α-synuclein

Abstract The pigmentation of substantia nigra pars compacta dopaminergic neurons is due to the presence of neuromelanin, an irregular macromolecular pigment belonging to the family of melanins. Depletion of neuromelanin in Parkinson’s disease is typically indicated by loss of brown color in this area. Unlike that from controls, the pigment extracted from substantia nigra of parkinsonian patients seems to be mainly composed by highly cross-linked, protease-resistant proteic material and the neuromelanin macromolecule appears to be a minor presence. In the present paper we describe the isolation by SDS-PAGE of this proteic component after cleavage of the melanin backbone under solubilizing conditions. A single band is observed, which has been identified as α-synuclein by western blotting. As expected, the same process performed on a control specimen did not show occurrence of any major proteic component. Nevertheless, extraction from a 91 years old control with Lewy bodies displayed minor α-synuclein immunoreactive aggregates, whereas inclusion of free α-synuclein was not observed at all. Results reported here support the view that α-synuclein accumulates within substantia nigra neurons and is entrapped in pigment granules during neuromelanin biosynthesis, i.e. before the melanin depletion characteristic of Parkinson’s disease starts.

Q-band EPR investigations of neuromelanin in control and Parkinson's disease patients

New insights into the understanding of the changes induced in the iron domain of neuromelanin (NM) upon development of Parkinsons disease (PD) have been gained by electron paramagnetic spectroscopy (EPR). The results of this study are compared with a previously reported variable temperature analysis of X-band EPR spectra of a NM specimen obtained from control brain tissues. The availability of high sensitivity instruments operating in the Q-band (34.4 GHz) allows us to deal with the low amounts of NM available from PD brains. The organization of iron in NM is in the form of polynuclear superparamagnetic/antiferromagnetic aggregates, but the lack of one or more signals in the EPR spectra of NM from PD suggests that the development of the pathology causes NM to decrease its ability to bind iron. Furthermore, the detection of the Mn(II) signal in the Q-band spectra is exploited as an additional internal probe to assess minor structural differences in iron domains of PD and control NM specimens.

Isolation and 13C-NMR characterization of an insoluble proteinaceous fraction from substantia nigra of patients with Parkinson's disease

Neuromelanin is a dark brown pigment suspected of being involved in the pathogenesis of Parkinsons disease. This pigment can be isolated from normal human substantia nigra by a procedure that includes an extensive proteolytic treatment. In this study we used such a procedure to extract the neuromelanin pigment from a pool of substantia nigra from patients affected by Parkinsons disease. 13C Cross polarization magic angle spinning nuclear magnetic resonance spectroscopy and electron paramagnetic resonance spectroscopy were used to characterize the solid residue obtained from the extraction procedure. We found that the pigment extracted from the substantia nigra of parkinsonian patients was mainly composed of highly cross‐linked, protease‐resistant, lipo‐proteic material, whereas the neuromelanin macromolecule appears to be only a minor component of this extract. A synthetic model of melanoprotein has been prepared by enzymatic oxidation of dopamine in the presence of albumin. Once it has undergone the same proteolytic treatment, this model system yields a 13C‐NMR spectrum which is similar to that observed for the parkinsonian midbrain extract. These results are consistent with the view that oxidative stress has a relevant role in the pathogenesis of Parkinsons disease.

Magnetic investigations of human mesencephalic neuromelanin

Pigmentation of neurons in substantia nigra is due to neuromelanin, a pigment that stores large amounts of iron. Human mesencephalic neuromelanin has been investigated by means of magnetic susceptibility measurements as a function of temperature. Magnetic measurements provide a physico-chemical characterization of the iron cluster buried in the organic melanin matrix and support the view that iron is not simply chelated, but rather is organized in a three-dimensional network. The paramagnetism of isolated iron ions is observed, in agreement with electron paramagnetic resonance spectroscopy. Furthermore, antiferromagnetic grains with a large size distribution function are present. These grains contain N spins coupled antiferromagnetically; however, N(1/2) spins are decoupled from the grain bulk and parallelly aligned. The latter subgrains are superparamagnetic with a blocking temperature ranging between 5 K and room temperature. This behavior has not been observed in synthetic melanin, where the paramagnetic contribution is strongly enhanced. Preliminary results on pigment isolated from patients affected by Parkinsons disease, a neurodegenerative pathology involving primarily pigmented neurons in substantia nigra pars compacta, show a lower total magnetization compared to control neuromelanin. The temperature behavior of zero field cooling and field cooling magnetizations is similar for both. The significant depletion of iron content in Parkinsons disease neuromelanin could indicate a progressive Fe migration from its storage environment to the cytosol.

Nuclear magnetic relaxation dispersion profiles of substantia nigra pars compacta in Parkinson's disease patients are consistent with protein aggregation

Nuclear Magnetic Relaxation field-cycling relaxometry is a technique, able to report on water mobility in tissues. By means of this technique, post-mortem specimens from both controls and idiopathic Parkinsons disease patients have been investigated. Results show different relaxometric behavior between the groups, which is consistent with protein aggregation in Parkinsons disease specimens.

Lymphocyte proteomics of Parkinson’s disease patients reveals cytoskeletal protein dysregulation and oxidative stress

AIMS There is increasing evidence of biochemical alterations in peripheral blood lymphocytes of Parkinsons disease (PD) patients. In this work, we describe the changes in protein levels in peripheral lymphocytes of PD patients in order to identify potential peripheral biomarkers. MATERIALS & METHODS By means of 2D electrophoresis and mass spectrometry protein identification, we compared patients under L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, patients under subthalamic nucleus deep-brain stimulation and healthy controls. RESULTS Statistical analysis of the results demonstrated that cofilin-1, tropomyosin, and a specific actin isoform vary significantly in patients, regardless of the therapy. Two different isoforms of gamma-fibrinogen either correlate with the disease state or with the disease duration. Eventually, specific changes associated with the different therapies allowed to highlight oxidative stress conditions in lymphocytes in patients treated with higher doses of L-DOPA. CONCLUSIONS As a whole, peripheral blood lymphocytes are sensitive reporters of PD over inter-individual variability, and allow the identification of specific alterations that could be further exploited for diagnostic purposes.

Proteome analysis of mesencephalic tissues: evidence for Parkinson's disease

Abstract.Proteome analysis is a powerful methodology to investigate protein expression in tissues involved in diseases not linked to particular genetic defects. To date, this technique has a limited number of applications in the field of neurodegenerative disorders. We decided therefore to investigate by this approach autoptic mesencephalic tissues of patients with idiopathic Parkinson’s disease as well as control specimens from healthy subjects.

A novel 19F-NMR method for the investigation of the antioxidant capacity of biomolecules and biofluids

A new assay for the measurement of the antioxidant capacity of biomolecules by high resolution 19F-NMR spectroscopy is presented here. This method is based on the use of trifluoroacetanilidic detectors, namely trifluoroacetanilide, N-(4-hydroxyphenyl)-trifluoroacetamide and 2-hydroxy-4-trifluoroacetamidobenzoic acid. Upon hydroxyl radical attack, such fluorinated detectors yield trifluoroacetamide and trifluoroacetic acid that can be quantitatively determined by 19F-NMR spectroscopy. Trifluoroacetamide was found to be a reliable reporter of hydroxyl radical attack on the fluorinated detectors, whereas N-(4-hydroxyphenyl)-trifluoroacetamide was found to be the most sensitive detector amongst the ones considered. Therefore, N-(4-hydroxyphenyl)-trifluoroacetamide has been used in competition experiments to assess the antioxidant capacity of a number of low and high molecular weight antioxidants. The antioxidant capacity of a given compound has been scaled in terms of an adimensional parameter, kF, that represents the ratio between the scavenger abilities of the fluorinated detector and the competitor. kF values obtained for low-molecular-mass compounds fall in the range 0.17 < kF < 1.5 and are in good agreement with second order rate constants (k2OH) for the reaction of the antioxidant with hydroxyl radicals. The kF value for serum albumin is much larger (46.9) than that predicted from the reported k2OH value. This finding supports the view that the protein can very effectively scavenge hydroxyl radicals as well as secondary radicals. Human blood serum showed that its antioxidant capacity is even higher than that shown by aqueous solutions of albumin at physiologic concentration suggesting a further contribution from other macromolecular serum components.