STochastic Optical Reconstruction Microscopy (STORM) reveals the nanoscale organization of pathological aggregates in human brain.

AIMS: Histological analysis of brain tissue samples provides valuable information about the pathological processes leading to common neurodegenerative disorders. In this context, the development of novel high-resolution imaging approaches is a current challenge in neuroscience. METHODS: To this end, we used a recent super-resolution imaging technique called STochastic Optical Reconstruction Microscopy (STORM) to analyse human brain sections. We combined STORM cell imaging protocols with neuropathological techniques to image cryopreserved brain samples from control subjects and patients with neurodegenerative diseases. RESULTS: This approach allowed us to perform 2D-, 3D- and two-colour-STORM in neocortex, white matter and brainstem samples. STORM proved to be particularly effective at visualizing the organization of dense protein inclusions and we imaged with a <50 nm resolution pathological aggregates within the central nervous system of patients with Alzheimer's disease, Parkinson's disease, Lewy body dementia and fronto-temporal lobar degeneration. Aggregated Aß branches appeared reticulated and cross-linked in the extracellular matrix, with widths from 60 to 240 nm. Intraneuronal Tau and TDP-43 inclusions were denser, with a honeycomb pattern in the soma and a filamentous organization in the axons. Finally, STORM imaging of α-synuclein pathology revealed the internal organization of Lewy bodies that could not be observed by conventional fluorescence microscopy. CONCLUSIONS: STORM imaging of human brain samples opens further gates to a more comprehensive understanding of common neurological disorders. The convenience of this technique should open a straightforward extension of its application for super-resolution imaging of the human brain, with promising avenues to current challenges in neuroscience.

Microdosimetry in 3D realistic mitochondria phantoms: Geant4 Monte Carlo tracking of 250keV photons in phantoms reconstructed from microscopic images.

Mitochondria are considered to be sensitive radiation targets since they control processes vital to the cell's functioning. These organelles are starting to get attention and some studies are investigating the radiation dose inside them. In previous studies, mitochondria are represented as simple ellipsoids inside the cell not taking into consideration the complexity of their shape. In this study, realistic phantoms are built based on deconvolved widefield fluorescent microscopic images of the mitochondrial networks of fibroblast cells. The phantoms are imported into Geant4 as tessellated volumes taking into account the geometrical complexity of these organelles. Irradiation with 250keV photons is performed and the lineal energy is calculated. The lineal energy distributions inside the produced phantoms are compared with those calculated inside simple volumes, a sphere and an ellipsoid, where the effect of the shape and volume is clearly seen on lineal energies.

Perspectives of drug-based neuroprotection targeting mitochondria.

Mitochondrial dysfunction has been reported in most neurodegenerative diseases. These anomalies include bioenergetic defect, respiratory chain-induced oxidative stress, defects of mitochondrial dynamics, increase sensitivity to apoptosis, and accumulation of damaged mitochondria with instable mitochondrial DNA. Significant progress has been made in our understanding of the pathophysiology of inherited mitochondrial disorders but most have no effective therapies. The development of new metabolic treatments will be useful not only for rare mitochondrial disorders but also for the wide spectrum of common age-related neurodegenerative diseases shown to be associated with mitochondrial dysfunction. A better understanding of the mitochondrial regulating pathways raised several promising perspectives of neuroprotection. This review focuses on the pharmacological approaches to modulate mitochondrial biogenesis, the removal of damaged mitochondria through mitophagy, scavenging free radicals and also dietary measures such as ketogenic diet.

Phenotypic spectrum of MFN2 mutations in the Spanish population.

INTRODUCTION: The most common form of axonal Charcot-Marie-Tooth (CMT) disease is type 2A, caused by mutations in the mitochondrial GTPase mitofusin 2 (MFN2). OBJECTIVE: The objective of our study is to establish the incidence of MFN2 mutations in a cohort of Spanish patients with axonal CMT neuropathy. MATERIAL AND METHODS: Eighty-five families with suspected axonal CMT were studied. All MFN2 exons were studied through direct sequencing. A bioenergetics study in fibroblasts was conducted using a skin biopsy taken from a patient with an Arg468His mutation. RESULTS: Twenty-four patients from 14 different families were identified with nine different MFN2 mutations (Arg94Trp, Arg94Gln, Ile203Met, Asn252Lys, Gln276His, Gly296Arg, Met376Val, Arg364Gln and Arg468His). All mutations were found in the heterozygous state and four of these mutations had not been described previously. MFN2 mutations were responsible for CMT2 in 16% +/- 7% of the families studied and in 30.8 +/- 14.2% (12/39) of families with known dominant inheritance. The bioenergetic studies in fibroblasts show typical results of MFN2 patients with a mitochondrial coupling defect (ATP/O) and an increase of the respiration rate linked to complex II. CONCLUSION: It is concluded that mutations in MFN2 are the most frequent cause of CMT2 in this region. The Arg468His mutation was the most prevalent (6/14 families), and our study confirms that it is pathological, presenting as a neuropathy in a mild to moderate degree. This study also demonstrates the value of MFN2 studies in cases of congenital axonal neuropathy, especially in cases of dominant inheritance, severe clinical symptoms or additional symptoms such as optic atrophy.

Mitochondrial activity in XTC.UC1 cells derived from thyroid oncocytoma.

Thyroid oncocytoma is characterized by the presence of oncocytes containing abnormally large numbers of mitochondria. However, the relationship between the abundance of mitochondria and the pathogenesis of the tumors is unknown. Recently, a new cell line, named XTC.UC1, has been derived from a metastasis of thyroid oncocytoma. We have studied the metabolism and the gene expression profile of the mitochondria in XTC.UC1 cells, using B-CPAP cells as controls. There were no signs of mitochondrial respiratory chain defects or uncoupling between the respiratory chain and adenosine triphosphate (ATP) production. In XTC.UC1 cells, mtDNA transcripts were increased more than fivefold than in controls, in parallel with a 3.6-fold increase in mtDNA content. Finally, in spite of the glycolytic metabolism induced by the culture medium, the mitochondria of XTC.UC1 cells possess the phenotype of oncocytic cells with hypertrophic mitochondria, higher respiratory enzyme activity and higher mtDNA content than in controls. XTC.UC1 cells may therefore offer a useful model for investigating the coordination of the nuclear and mitochondrial genomes, in the context of thyroid tumors.

Induction of ANT2 gene expression in liver of patients with mitochondrial DNA depletion.

We have previously described two cases of children with a liver mitochondrial DNA (mtDNA) depletion syndrome, characterised by a low ratio of mtDNA to nuclear DNA. Light microscopy performed on liver biopsy showed abnormal hepatocytes with a characteristic 'oncocytic' appearance, indicative of perturbed oxidative phosphorylation. The adenine nucleotide translocator (ANT), the last step in oxidative phosphorylation catalyses the exchange of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) between the cytosol and mitochondria. The ANT2 gene, which is not normally expressed in human tissues, encodes an isoform preferentially expressed under conditions of glycolytic metabolism. ANT2 gene expression is regulated by a transcription factor involved in a molecular mechanism selecting for the import of glycolytic ATP into the mitochondrial matrix. This ATP import is required in highly proliferative cells, such as tumour cells, which are highly dependent on glycolysis for ATP synthesis. We postulated that, as a result of the defective oxidative phosphorylation observed in these patient biopsies, the ANT2 expression would be induced. We simultaneously quantified the mtDNA depletion and the ANT2 gene expression in liver biopsies from the two patients and six controls. ANT2 mRNA levels were significantly increased in the two patient liver biopsies. Moreover, in one patient, the liver mtDNA depletion was found to be partially reversed after less than 4 years and this reversion was coupled to a concomitant decrease of the ANT2 expression. These results suggest that dysfunction of oxidative phosphorylation could lead to a switch from mitochondrial to glycolytic ATP production, to restore tissue-specific energy requirements.