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.

Cognitive deficit, and neuropathological correlates, in the oldest-old.

Several disorders are usually involved in the cognitive deficit of the oldest old. Alzheimer disease is the commonest. It is usually characterized by progressive memory impairment - neocortical symptoms occurring much later in the course of the disease. Alzheimer disease should not be considered any more as the single cause of a cognitive deficit in a very old patient. Vascular alterations, possibly causing microinfarcts, are commonly associated, especially in cerebral amyloid angiopathy. A slowly progressive memory deficit with negative CSF biomarkers of Alzheimer's disease may be due to hippocampal sclerosis that may be the consequence of multiple causes: in most of the cases, it is associated with neuronal TDP-43 inclusions. Recently, a distribution of these inclusions to a territory more extensive than the hippocampus has been reported and attributed to a new entity, called Limbic-predominant Age-related TDP-43 Encephalopathy (LATE) with or without hippocampal sclerosis. The presence of cortical Lewy bodies may cause an intellectual deficit or contribute to it. The prevalence of dementia with cortical Lewy bodies in the oldest old is discussed. Tau inclusions in cortical glia have also been shown to participate to the intellectual deficit. Association of neurodegenerative and vascular changes is the most frequent situation in the very old patients. Systemic diseases such as diabetes or heart failure, prescription drugs (when misused), or toxic such as alcohol may also contribute to the cognitive impairment and be amenable to treatment.

[Spreading of protein misfolding: A new paradigm in neurology]. / La transconformation protéique, nouveau paradigme en neurologie.

Protein misfolding and spreading ("transconformation") are being better understood. Described in Prions diseases, this new paradigm in the field of neurodegenerative disorders and brain aging also implies sporadic inclusion myositis, type 2 diabetes, some cancers, sickle cell disease... Misfolding is transmitted from a protein or peptide to a normally folded one. Often associated with a stress of the endoplasmic reticulum, it may spread along the neurites, following anterograde or retrograde axonal transport. In the central nervous system, it occurs in a few cells and there is invasion of adjacent cells by cell-to-cell spread. Three varieties of protein misfolding occur along neuroanatomical pathways. It can be a 'centripetal' process. The synucleinopathy of Parkinson disease has been carefully studied: the changes first occur in cardiac or enteric plexuses... and reach later on the mesencephalon and neocortex. Thus, skin biopsy might prove a diagnostic tool. Protein misfolding may also occur along 'centrifugal' pathways, from motor cortex to peripheral motor neurons. Examples are provided by SOD and pTDP-43 in Amyotrophic Lateral Sclerosis. Amyloid ß peptide in cerebral aging and Alzheimer's disease also spread from occipital cortex to the brainstem. Lastly, the propagation may remain 'central' for TDP-43 in behavioral variant frontotemporal dementia, following only pathways of the encephalic neural network. This has to be confirmed, however, since the spreading of some proteins (such as tau or Aß peptides) has been considered central for a long time and has proved today to involve extracerebral tissues. The complex mechanisms of protein misfolding, still in analysis, include the involvement of chaperone proteins, the formation of very toxic labile proteins molecules (oligomers?), and provide a number of new therapeutic perspectives.

Neurodegenerative lesions: seeding and spreading.

Accumulation of specific proteins has replaced loss of specific populations of neurons in the definition of most neurodegenerative diseases. In some cases, the amino-acid sequence of the protein that accumulates is altered by a mutation in the gene that codes for it but most generally, the primary structure is normal. Much evidence from human neuropathology has been collected over the years indicating that the progression of the lesions in such neurodegenerative diseases as Alzheimer's disease, Parkinson's disease and progressive supranuclear palsy follow the neuroanatomical connections. More recently, injection of aggregates of the specific proteins in the brain of experimental animals has been attempted in various experimental settings. Brain homogenates containing Aß aggregates induce the early development of Aß deposits in APP transgenic mice. Brain homogenates from various human tauopathies induce tau aggregates in transgenic mice expressing normal human tau. Finally, synthetic preformed fibrils of alpha-synuclein initiate the development of alpha-synuclein accumulation resembling Parkinson's disease in wild-type mice. Experiments in cell cultures suggest that the protein has to be in some specific state of oligomerization or fibrillation to be endocytosed and transported by the neuron. These data suggest that the protein that accumulates in a specific disease is initially misfolded and that this misfolding contaminates normal protein in a prion-like manner - in some cases through the neuronal connections.

Frontotemporal lobar degeneration: diversity of FTLD lesions.

Frontotemporal lobar degeneration (FTLD) is a heterogeneous group including both sporadic and familial diseases, characterized by a macroscopic alteration. It may correspond to various cognitive syndromes: behavioral variant of frontotemporal dementia (bvFTD), progressive nonfluent aphasia, and semantic dementia. The neuropathologic classification is now based on identification of the protein that accumulates in neurons and glia: Tau, TAR DNA Binding Protein 43 (TDP-43), and FUsed in Sarcoma (FUS). The disorders in which the corresponding proteins accumulate have been named FTLD-Tau, FTLD-TDP, and FTLD-FUS. FTLD-Tau includes sporadic cases (e.g. Pick's disease) and Tau mutations. FTLD-TDP are subdivided within four types (A, B, C, D) according to the shape and distribution of TDP-43 positive lesions within the associative frontal cortex. The FTLD-FUS group includes atypical FTLD with ubiquitinated lesions (FTLD-U), Neuronal Intermediate Filament Inclusion Disease (NIFID) and Basophilic Inclusion Body Disease (BIBD).

Induction and characterization of mitochondrial DNA mutants in Chlamydomonas reinhardtii.

In addition to lethal minute colony mutations which correspond to loss of mitochondrial DNA, acriflavin induces in Chlamydomonas reinhardtii a low percentage of cells that grow in the light but do not divide under heterotrophic conditions. Two such obligate photoautotrophic mutants were shown to lack the cyanide-sensitive cytochrome pathway of the respiration and to have a reduced cytochrome c oxidase activity. In crosses to wild type, the mutations are transmitted almost exclusively from the mating type minus parent. A same pattern of inheritance is seen for the mitochondrial DNA in crosses between the two interfertile species C. reinhardtii and Chlamydomonas smithii. Both mutants have a deletion in the region of the mitochondrial DNA containing the apocytochrome b gene and possibly the unidentified URFx gene.

[Morphologic and molecular neuropathology of Alzheimer's disease]. / Neuropathologie morphologique et moléculaire de la maladie d'Alzheimer.

Alzheimer disease lesions include the abnormal accumulation of two proteins normally present in neurons: tau protein and Abeta peptide. Tau protein aggregates into fibrils in the cell body of neurons (neurofibrillary tangles), in dendrites (neuropil threads) and in degenerating axons that constitute the corona of the senile plaque. Tau pathology progresses in the brain areas in a stereotyped manner and in parallel with the clinical symptoms. Abeta extracellular deposits may be diffuse or focal. The Abeta focal deposit constitutes the core of the senile plaque. Progression of the Abeta lesions, which initially affect the isocortex, then the hippocampus, basal ganglia, various brainstem nuclei and cerebellum, is not directly correlated with symptoms. Mutations involving the genes implicated in Abeta peptide metabolism are responsible for familial Alzheimer disease. Mutations of the tau gene are not associated with Alzheimer disease but with frontotemporal dementia. The link between altered Abeta peptide metabolism and tau pathology has not been fully elucidated. Animal models mimic several aspects of the disease and have contributed to a better understanding of the mechanisms of the lesions.

[Nosology of dementias: the neuropathologist's point of view]. / Nosologie des démences. Le point de vue du neuropathologiste.

The diagnosis of degenerative dementias heavily relies on the identification of neuronal or glial inclusions. Tauopathy is probably the largest group including Alzheimer and Pick disease, mutation of the tau gene, progressive supranuclear palsy, corticobasal degeneration, and argyrophilic grain disease. Lewy bodies, when numerous in the cerebral cortex, are usually associated with the cognitive deficit of Parkinson disease dementia or of dementia with Lewy bodies--both conditions being distinguished by clinical information. The inclusions of the dentate gyrus, only labeled by anti-ubiquitin antibodies, isolate a subgroup of fronto-temporal dementia (FTDu), sometimes familial and sometimes associated with amyotrophic lateral sclerosis. Mutations of the progranulin gene have been recently discovered among a significant proportion of these patients. Neuronal Intermediate Filament Inclusion Disease (NIFID) is a rare, apparently sporadic dementia, characterized by the presence of large inclusions in the cell body of many neurons. These inclusions react with antibodies directed against neurofilaments or against other intermediate filaments (such as alpha-internexin). The diagnostic value of some of these inclusions allowing the classification of the degenerative dementias has been discussed. The link between the inclusions and the pathogenetic mechanism is indeed probably variable. It should however be stressed that whenever their composition has been elucidated, the inclusions have given important clues to the pathogenesis of the disease in which they had been found.

Kinetic study of the aspartate/glutamate carrier in intact rat heart mitochondria and comparison with a reconstituted system.

The homologous exchange of external [14C] aspartate/internal aspartate catalyzed by the aspartate/glutamate carrier of rat heart mitochondria was investigated using aspartate-loaded, glutamate-depleted mitochondria. An inhibitor-stop technique was developed for kinetic studies by applying pyridoxal phosphate. Direct initial rate determinations from the linear phase of [14C] aspartate uptake were insufficiently accurate at high external and/or low internal substrate concentrations. Therefore, the full time-course of [14C] aspartate uptake until reaching isotope equilibrium was fitted by a single exponential function and was used to calculate reliable initial steady-state rates. This method was applied in bisubstrate analyses of the antiport reaction for different external and internal aspartate concentrations. The kinetic patterns obtained in double reciprocal plots showed straight lines converging on the abscissa. This result is consistent with a sequential antiport mechanism. It implies the existence of a catalytic ternary complex that is formed by the translocator and substrate molecules bound from both sides of the membrane. The Km values for aspartate were clearly different for the external and the internal sides of the membrane, 216 +/- 23 microM and 2.4 +/- 0.5 mM, respectively. These values indicated a definite transmembrane asymmetry of the carrier. The same asymmetry became evident when investigating the isolated protein from bovine heart mitochondria after reconstitution into liposomes. In this case the Km values for external and internal aspartate were determined to be 123 +/- 11 microM and 2.8 +/- 0.6 mM, respectively. This comparison demonstrates a right-side out orientation of the carrier after insertion into liposomal membranes. The sequential transport mechanism of the aspartate/glutamate carrier, elucidated both in proteoliposomes and in mitochondria, also seems to be a common characteristic of other mitochondrial antiport carriers.

Effect of aspartate and glutamate on the oxoglutarate carrier investigated in rat heart mitochondria and inverted submitochondrial vesicles.

Interaction of glutamate and aspartate with the oxoglutarate carrier was investigated in rat heart mitochondria or inverted submitochondrial particles. With mitochondria, glutamate and aspartate had no effect on the initial rate of oxoglutarate or malate uptake. With inverted submitochondrial vesicles, binding experiments indicated that aspartate bound to the oxoglutarate carrier on its matricial face and increased the affinity of the substrate binding site for malate but did not change the affinity for oxoglutarate. Glutamate had no effect on both substrate bindings. The dissociation constants of the binary substrate-carrier complexes on the matricial side were determined (1.28 +/- 0.15 mM for oxoglutarate and 2.22 +/- 0.26 mM for malate). These values, compared with those obtained previously on the cytosolic side of intact mitochondria, confirmed the asymmetry of the carrier in the native membrane (higher affinities on the cytosolic face). It is concluded that (1) aspartate and glutamate are not cytosolic effectors of the oxoglutarate carrier, (2) matricial aspartate is a positive effector of the binding of malate on the matricial side of the oxoglutarate carrier, and (3) such a characteristic may play a role in the regulation of the oxoglutarate carrier. Thus, it may be emphasized that (1) this observation is the first clear evidence of a well-defined 'sophisticated regulation' (allosteric) of a mitochondrial metabolite carrier, and (2) this regulation of the oxoglutarate carrier may have important consequences on the efficiency of reducing equivalent import in the matrix space by the malate-aspartate shuttle.

Sicca syndrome and dementia in a patient with hepatitis C infection: a case report with unusual bifocal extrahepatic manifestations.

INTRODUCTION: Hepatitis C virus (HCV) infections are associated with extrahepatic manifestations in 40-75 % of cases. Sialitis and secondary Sjögren syndrome are well characterized complications of chronic HCV infections but the mechanisms (primary or secondary) leading to xerostomia are not understood. Similarly, brain lesions due to HCV can be primary or secondary but the pathology of primary HCV-related brain lesions is not well described. CASE REPORT: We report the postmortem case of a 60-year old patient initially presenting with sicca syndrome and dementia. HCV was identified in the brain but not in the salivary glands using transcription-mediated amplification (TMA). Focal sialitis was found in submandibular glands. Neuropathological examination revealed the presence of multiple dot-sized demyelination foci. CONCLUSION: Sicca syndrome is a common concern in chronic HCV infections and may be due to secondary immune mechanisms (we could not isolate HCV in salivary gland tissues). TMA had never been applied to the detection of viruses in salivary glands and neural tissues and proves to be a promising technique. Neuropathological reports in HCV infections are rare and the lesions we report may be the first characterization of the direct effect of HCV on brain cells. More cases are needed to define the full spectrum of lesions potentially caused by the direct action of the HCV on salivary glands and neural tissues.

Modifications of the endosomal compartment in peripheral blood mononuclear cells and fibroblasts from Alzheimer's disease patients.

Identification of blood-based biomarkers of Alzheimer's disease (AD) remains a challenge. Neuropathological studies have identified enlarged endosomes in post-mortem brains as the earliest cellular change associated to AD. Here the presence of enlarged endosomes was investigated in peripheral blood mononuclear cells from 48 biologically defined AD patients (25 with mild cognitive impairment and 23 with dementia (AD-D)), and 23 age-matched healthy controls using immunocytochemistry and confocal microscopy. The volume and number of endosomes were not significantly different between AD and controls. However, the percentage of cells containing enlarged endosomes was significantly higher in the AD-D group as compared with controls. Furthermore, endosomal volumes significantly correlated to [C(11)]PiB cortical index measured by positron emission tomography in the AD group, independently of the APOE genotype, but not to the levels of amyloid-beta, tau and phosphorylated tau measured in the cerebrospinal fluid. Importantly, we confirmed the presence of enlarged endosomes in fibroblasts from six unrelated AD-D patients as compared with five cognitively normal controls. This study is the first, to our knowledge, to report morphological alterations of the endosomal compartment in peripheral cells from AD patients correlated to amyloid load that will now be evaluated as a possible biomarker.

Office of Rare Diseases neuropathologic criteria for corticobasal degeneration.

A working group supported by the Office of Rare Diseases of the National Institutes of Health formulated neuropathologic criteria for corticobasal degeneration (CBD) that were subsequently validated by an independent group of neuropathologists. The criteria do not require a specific clinical phenotype, since CBD can have diverse clinical presentations, such as progressive asymmetrical rigidity and apraxia, progressive aphasia, or frontal lobe dementia. Cortical atrophy, ballooned neurons, and degeneration of the substantia nigra have been emphasized in previous descriptions and are present in CBD, but the present criteria emphasize tau-immunoreactive lesions in neurons, glia, and cell processes in the neuropathologic diagnosis of CBD. The minimal pathologic features for CBD are cortical and striatal tau-positive neuronal and glial lesions, especially astrocytic plaques and thread-like lesions in both white matter and gray matter, along with neuronal loss in focal cortical regions and in the substantia nigra. The methods required to make this diagnosis include histologic stains to assess neuronal loss, spongiosis and ballooned neurons, and a method to detect tau-positive neuronal and glial lesions. Use of either the Gallyas silver staining method or immunostains with sensitive tau antibodies is acceptable. In cases where ballooned neurons are sparse or difficult to detect, immunostaining for phospho-neurofilament or alpha-B-crystallin may prove helpful. Methods to assess Alzheimer-type pathology and Lewy body pathology are necessary to rule out other causes of dementia and Parkinsonism. Using these criteria provides good differentiation of CBD from other tauopathies, except frontotemporal dementia and Parkinsonism linked to chromosome 17, where additional clinical or molecular genetic information is required to make an accurate diagnosis.

Diagnosis and staging of Alzheimer disease.

Rather than determining lesions "threshold" between "normal" cases and patients, we prefer to use clinicopathological correlations, assigning a given intellectual deficit to a given amount of lesions with a chosen level of probability. Because large amounts of A beta diffuse deposits may be found in the absence of dementia, we think advisable not to take them into account for the diagnosis. The diffusion of the neurofibrillary tangles in the paralimbic, limbic and isocortical areas (described by braak and Braak stages or by the number of areas containing tangles) and the density of isocortical senile plaques (A beta focal deposits) as assessed by the CERAD protocol are both correlated with the intellectual status but give complementary information. They should thus be jointly used. We analyzed the variability of the lesions counts, their coefficients of error, and their causes, as a first step toward standardization. We have shown, however, that semiquantitative estimates are presently more reproducible than quantitative measures.

Beta A4 deposits are constant in the brain of the oldest old: an immunocytochemical study of 20 French centenarians.

beta A4 deposits occur in the brain of some individuals over 50 years of age. It could be a part of the aging process or indicate a disease found frequently in the elderly. To address this question, beta A4 immunocytochemistry was performed on the brain of 15 nondemented and 5 demented centenarians, some of whom were affected by Alzheimer's disease. We found beta A4 deposits in the parahippocampal and the superior temporal gyri of all the cases, whatever the clinical state. The hippocampus was frequently spared. The lesion density was not correlated with the severity of the mental deterioration. The constant deposition of beta A4 protein in the brain of very old people indicates that this process does not spare a large proportion of this population. This result favors beta A4 accumulation in the brain being an ineluctable age-related process.

Microglia, amyloid and dementia in alzheimer disease. A correlative study.

To elucidate the role of microglia in Alzheimer's disease, a clinicopathological study was performed involving 26 cases, the mental status of which had been studied pre mortem by the Blessed test score (BTS). We measured the volume density of CD 68 immunoreactive (IR) microglia, congophilic plaques and Abeta deposits, and the numerical density of neurofibrillary tangles (NFT) in a sample of Area 9 (middle frontal gyrus). Dementia was significantly correlated only with the volume density of Abeta deposits and the numerical density of NFT. The volume densities of microglia and congophilic plaques were strongly correlated. With the intellectual status used as a time scale, IR microglia and amyloid deposits appeared almost simultaneously at an early stage in the pathological cascade and decreased, whereas Abeta and NFT were still accumulating. The intellectual deficit seemed to be more significantly related to the latter two lesions than to the microglia-amyloid complex, that was visible at an earlier stage (around BTS = 15).

MRI description of cerebral atrophy in mouse lemur primates.

We assessed cerebral atrophy in mouse lemur primates (Microcebus murinus) by estimating CSF volume in their brains from 4.7 Tesla T2-weighted magnetic resonance images. Thirty animals aged from 1 to 10.3 years were imaged, 14 of them were followed for up to 2 years. Seven of these animals were examined for neuropathology. In 12 out of 17 animals older than 3.5 years, CSF volumes were increased. A subgroup of six animals had severe atrophy of the temporal lobe. Another subgroup of five animals displayed diffuse atrophy in addition to the temporal atrophy. One animal had a dilation of the external part of the temporal horn of the lateral ventricle in addition to the temporal atrophy. The three animals with diffuse atrophy that could be studied for neuropathology had diffuse cerebral amyloid deposits detected by immunocytochemistry. The other animals did not display amyloid deposits. Relations between the different types of atrophy as well as their causes will have to be assessed in future studies.

Modeling the relation between neurofibrillary tangles and intellectual status.

The relationship between the neurofibrillary tangles and the intellectual deficit observed in senile dementia of the Alzheimer type was studied in 27 patients over the age of 75. The presence and density of tau positive tangles were assessed in six areas including limbic, paralimbic, and isocortical cortices. In the isocortical areas, the presence [1] or absence [0] of neurofibrillary tangles was better correlated with the Blessed test score than the density of the neurofibrillary tangles profiles. Multivariate analysis showed that the number of areas containing at least one neurofibrillary tangle was the best explanatory variable of the intellectual status. The cortical areas were ranked according to the prevalence of their involvement. The presence of tangles in an area of a given rank took place only if the areas of lower ranks were also involved. It is proposed that the presence of tangles in a given area is a more significant information than the value of their density. These data may lead to new diagnostic procedures.

Cerebral T2-weighted signal decrease during aging in the mouse lemur primate reflects iron accumulation.

4.7 Tesla T2-weighted magnetic resonance images showed a highly significant signal decrease in the pallidum, substantia nigra, putamen, and a less significant decrease in the thalamus and the caudate of aging mouse lemurs (Microcebus murinus). We evaluated the contribution of iron deposits to the signal decrease comparing Perls' stained histological sections of six mouse lemurs brains aged 1 to 10 years to magnetic resonance images. In young animals, none of the brain structures was stained. A large number of iron deposits were visible in the pallidum and substantia nigra of aged animals and a moderate number in the middle aged ones. In the putamen, few iron deposits were visible in aged and middle-aged animals. The thalamus and the caudate appeared unstained with Perls' technique; iron was too low to be detected. The intensification of the reaction by diaminobenzidine revealed iron deposits in the thalamus of aging animals. This study suggests that in mouse lemurs, iron deposits are responsible for T2-weighted signal decrease in the central gray nuclei.