Stochastic Optical Reconstruction Microscopy Imaging of Multiple System Atrophy Inclusions Suggests Stepwise α-Synuclein Aggregation.

BACKGROUND: The architecture and composition of glial (GCI) and neuronal (NCI) α-synuclein inclusions observed in multiple system atrophy (MSA) remain to be precisely defined to better understand the disease. METHODS: Here, we used stochastic optical reconstruction microscopy (STORM) to characterize the nanoscale organization of glial (GCI) and neuronal (NCI) α-synuclein inclusions in cryopreserved brain sections from MSA patients. RESULTS: STORM revealed a dense cross-linked internal structure of α-synuclein in all GCI and NCI. The internal architecture of hyperphosphorylated α-synuclein (p-αSyn) inclusions was similar in glial and neuronal cells, suggesting a common aggregation mechanism. A similar sequence of p-αSyn stepwise intracellular aggregation was defined in oligodendrocytes and neurons, starting from the perinuclear area and growing inside the cells. Consistent with this hypothesis, we found a higher mitochondrial density in GCI and NCI compared to oligodendrocytes and neurons from unaffected donors (P < 0.01), suggesting an active recruitment of the organelles during the aggregation process. CONCLUSIONS: These first STORM images of GCI and NCI suggest stepwise α-synuclein aggregation in MSA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Transactive response DNA-binding protein 43 is enriched at the centrosome in human cells.

The centrosome, as the main microtubule organizing centre, plays key roles in cell polarity, genome stability and ciliogenesis. The recent identification of ribosomes, RNA-binding proteins and transcripts at the centrosome suggests local protein synthesis. In this context, we hypothesized that TDP-43, a highly conserved RNA binding protein involved in the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, could be enriched at this organelle. Using dedicated high magnification sub-diffraction microscopy on human cells, we discovered a novel localization of TDP-43 at the centrosome during all phases of the cell cycle. These results were confirmed on purified centrosomes by western blot and immunofluorescence microscopy. In addition, the co-localization of TDP-43 and pericentrin suggested a pericentriolar enrichment of the protein, leading us to hypothesize that TDP-43 might interact with local mRNAs and proteins. Supporting this hypothesis, we found four conserved centrosomal mRNAs and 16 centrosomal proteins identified as direct TDP-43 interactors. More strikingly, all the 16 proteins are implicated in the pathophysiology of TDP-43 proteinopathies, suggesting that TDP-43 dysfunction in this organelle contributes to neurodegeneration. This first description of TDP-43 centrosomal enrichment paves the way for a more comprehensive understanding of TDP-43 physiology and pathology.

Dedifferentiated cells obtained from glioblastoma cell lines are an easy and robust model for mesenchymal glioblastoma stem cells studies.

Glioblastoma is an aggressive brain tumor with a poor prognosis. Glioblastoma Stem Cells (GSC) are involved in glioblastoma resistance and relapse. Effective glioblastoma treatment must include GSC targeting strategy. Robust and well defined in vitroGSC models are required for new therapies evaluation. In this study, we extensively characterized 4 GSC models obtained by dedifferentiation of commercially available glioblastoma cell lines and compared them to 2 established patient derived GSC lines (Brain Tumor Initiating Cells). Dedifferentiated cells formed gliospheres, typical for GSC, with self-renewal ability. Gene expression and protein analysis revealed an increased expression of several stemness associated markers such as A2B5, integrin α6, Nestin, SOX2 and NANOG. Cells were oriented toward a mesenchymal GSC phenotype as shown by elevated levels of mesenchymal and EMT related markers (CD44, FN1, integrin α5). Dedifferentiated GSC were similar to BTIC in terms of size and heterogeneity. The characterization study also revealed that CXCR4 pathway was activated by dedifferentiation, emphasizing its role as a potential therapeutic target. The expression of resistance-associated markers and the phenotypic diversity of the 4 GSC models obtained by dedifferentiation make them relevant to challenge future GSC targeting therapies.

Design and application of a customizable relational DataBase to assess clinicopathological correlations and concomitant pathology in neurodegenerative diseases.

The diagnosis of neurodegenerative diseases is made complex by the heterogenous phenotype of the patients and the regular occurrence of concomitant pathology. Studying clinicopathological correlations in autopsy series is a central approach to improve pathological prediction in clinical practice. However, such method requires a wealth of information, and the use of standard spreadsheet software is hardly suitable. To overcome this constraint, we designed a customizable and freely available neuropathology form with 456 data entry fields driven by an open-source DataBase Management Systems (DBMS) using Structured Query Language (SQL). This approach allowed us to optimize the compilation of clinical and pathological data from our brain collection (264 autopsied patients, 22,885 data points). Information was then easily retrieved using general and specific queries, facilitating the analysis of demographics, clinicopathological correlations, and incidental and concomitant proteinopathies. Tau, amyloid-ß and α-synuclein incidental pathology was observed in respectively 78.1%, 42.8%, and 10.7% of all the patients. These proportions increased with age, reaching 100% for Tau pathology after 80. Concomitant proteinopathy was observed in 46.4% of the patients diagnosed with neurodegenerative diseases and prion disease. We observed a particularly high rate of co-pathology in patients with Dementia with Lewy bodies (81.3% of associated Tau and amyloid-ß pathology) and Creutzfeldt-Jakob disease (68.4% of associated Tau pathology). Finally, we used specific queries to identify old cases that could meet newly defined neuropathological criteria and revised the diagnosis of a 90-year-old patient to LATE Stage 2. Increasing our understanding of clinicopathological correlations in neurodegenerative diseases is crucial given the implications in clinical diagnosis, biomarker identification and targeted therapies assessment. The precise characterization of clinical and pathological data of autopsy series remains a central approach but the large amount of generated data should encourage a more systematic use of DBMS.

Glutamate-Induced Deregulation of Krebs Cycle in Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-Like Episodes (MELAS) Syndrome Is Alleviated by Ketone Body Exposure.

(1) Background: The development of mitochondrial medicine has been severely impeded by a lack of effective therapies. (2) Methods: To better understand Mitochondrial Encephalopathy Lactic Acidosis Syndrome Stroke-like episodes (MELAS) syndrome, neuronal cybrid cells carrying different mutation loads of the m.3243A > G mitochondrial DNA variant were analysed using a multi-omic approach. (3) Results: Specific metabolomic signatures revealed that the glutamate pathway was significantly increased in MELAS cells with a direct correlation between glutamate concentration and the m.3243A > G heteroplasmy level. Transcriptomic analysis in mutant cells further revealed alterations in specific gene clusters, including those of the glutamate, gamma-aminobutyric acid pathways, and tricarboxylic acid (TCA) cycle. These results were supported by post-mortem brain tissue analysis from a MELAS patient, confirming the glutamate dysregulation. Exposure of MELAS cells to ketone bodies significantly reduced the glutamate level and improved mitochondrial functions, reducing the accumulation of several intermediate metabolites of the TCA cycle and alleviating the NADH-redox imbalance. (4) Conclusions: Thus, a multi-omic integrated approach to MELAS cells revealed glutamate as a promising disease biomarker, while also indicating that a ketogenic diet should be tested in MELAS patients.

Improved detection of mitochondrial DNA instability in mitochondrial genome maintenance disorders.

PURPOSE: Diseases caused by defects in mitochondrial DNA (mtDNA) maintenance machinery, leading to mtDNA deletions, form a specific group of disorders. However, mtDNA deletions also appear during aging, interfering with those resulting from mitochondrial disorders. METHODS: Here, using next-generation sequencing (NGS) data processed by eKLIPse and data mining, we established criteria distinguishing age-related mtDNA rearrangements from those due to mtDNA maintenance defects. MtDNA deletion profiles from muscle and urine patient samples carrying pathogenic variants in nuclear genes involved in mtDNA maintenance (n = 40) were compared with age-matched controls (n = 90). Seventeen additional patient samples were used to validate the data mining model. RESULTS: Overall, deletion number, heteroplasmy level, deletion locations, and the presence of repeats at deletion breakpoints were significantly different between patients and controls, especially in muscle samples. The deletion number was significantly relevant in adults, while breakpoint repeat lengths surrounding deletions were discriminant in young subjects. CONCLUSION: Altogether, eKLIPse analysis is a powerful tool for measuring the accumulation of mtDNA deletions between patients of different ages, as well as in prioritizing novel variants in genes involved in mtDNA stability.

Metabolomic Sexual Dimorphism of the Mouse Brain is Predominantly Abolished by Gonadectomy with a Higher Impact on Females.

The importance of sexual dimorphism of the mouse brain metabolome was recently highlighted, in addition to a high regional specificity found between the frontal cortex, the cerebellum, and the brain stem. To address the origin of this dimorphism, we performed gonadectomy on both sexes, followed by a metabolomic study targeting 188 metabolites in the three brain regions. While sham controls, which underwent the same surgical procedure without gonadectomy, reproduced the regional sexual dimorphism of the metabolome previously identified, no sex difference was identifiable after gonadectomy, through both univariate and multivariate analyses. These experiments also made it possible to identify which sex was responsible for the dimorphism for 35 metabolites. The female sex contributed to the difference for more than 80% of them. Our results show that gonads are the main contributors to the brain sexual dimorphism previously observed, especially in females.

Late-onset camptocormia caused by a heterozygous in-frame CAPN3 deletion.

Camptocormia is defined by a pathological involuntary flexion of the thoracic and lumbar spine that is fully reducible in the supine position. Although originally described as a manifestation of conversion disorder, it is more commonly caused by a wide range of neurological diseases, in particular movement and neuromuscular disorders. We describe here a rare case of late onset camptocormia caused by autosomal dominant calpainopathy due to a heterozygous in-frame deletion in CAPN3 leading to loss of a single lysin amino acid in the catalytic domain of calpain-3. Creatine kinase levels, electromyography, and thigh muscle MRI were normal. Muscle biopsy did not show lobulated fibers and calpain-3 protein expression was not decreased, but in vitro functional assays showed impaired proteolytic function of. Lys254del CAPN3. Autosomal dominant calpainopathy should be considered in the differential diagnosis of late onset camptocormia and unexplained paravertebral myopathies even in presence of normal creatine kinase levels, and in absence of lobulated fibers, of decreased calpain-3 protein expression, and of muscle limb involvement.

Differences in clinical features between small fiber and sensitive large fiber neuropathies in Sjögren's syndrome.

BACKGROUND: To distinguish large (LFN) and small fiber neuropathies (SFN) in Sjögren's syndrome (SS) requires electroneuromyography (EMG) first, but this is time-consuming and has sometimes a limited accessibility, which can lead to a diagnostic delay. We aimed to identify clinical features that could distinguish SFN from sensitive LFN in SS. METHODS: The study included patients with SS who were monitored in the internal medicine and neurology departments at Angers University Hospital between 2010 and 2016, and who were tested for suspected peripheral neuropathy. Patients with clinical motor involvement were excluded. LFN diagnosis was based on EMG. SFN diagnosis was based on intraepidermal nerve fiber density on skin biopsies in patients with no abnormality on EMG. RESULTS: LFN and SFN were diagnosed respectively in 22 (6.9%) and 17 (5.4%) patients among 317 patients with SS. Prevalence of anti-SSA antibodies was lower in the SFN group compared to the LFN group (p=0.002). The types of paresthesia did not differ between the 2 groups. After adjustment for age and sex, SFN was associated with dysautonomia (p=0.01, OR 8.4 [CI 95%: 1.7-42.4]) and without length-dependent topography (p=0.03, OR 0.2 [0.04-0.8] in comparison with the LFN group. CONCLUSIONS: An association of non-length-dependent pattern and dysautonomia seems to predict the absence of LFN in SS and encourages the search for SFN. In contrary, patients with length-dependent involvement and without dysautonomia should be prioritized for EMG.

Metabolomics reveals highly regional specificity of cerebral sexual dimorphism in mice.

The development of personalized medicine according to gender calls for the integration of sexual dimorphism in pre-clinical models of diseases. Although sexual dimorphism in the brain of the mouse has been the subject of several behavioral, neuroimaging and experimental studies, very few have characterized the bases of sexual dimorphism in the brain on the omics scale. In particular, physiological variations in metabolomic and lipidomic terms related to gender have not been mapped in the brain. We carried out a metabolomic analysis, targeting 188 metabolites representative of various cellular structures and metabolisms, in three brain regions: frontal cortex, brain stem and cerebellum, in 3-month-old C57BL-6 J male (n = 20) vs. female (n = 20) mice. Our results demonstrate the existence of sexual dimorphism in the whole brain as well as in separate brain regions. Half of the 129 accurately measured metabolites were involved in the sexual dimorphism of the murine brain, but only 8% of those (hydroxyproline, creatinine, hexoses, tryptophan, threonine and lysoPC.a.C18.2) were involved in common in the three cerebral regions, while 71%, including phosphatidylcholines, lysophosphatidylcholines, sphingomyelins, acylcarnitines, amino acids, biogenic amines, and polyamines, were specific to only one region of the brain, underscoring the highly regional specificity of cerebral sexual dimorphism in mice.

Neurofilaments form flexible bundles during neuritogenesis in culture and in mature axons in situ.

Neurofilaments (NFs) undergo cation-dependent phospho-mediated associations with each other and other cytoskeletal elements that support axonal outgrowth. Progressive NF-NF associations generate a resident, bundled population that undergoes exchange with transporting NFs. We examined the properties of bundled NFs. Bundles did not always display a fully linear profile but curved and twisted at various points along the neurite length. Bundles retracted faster than neurites and retracted bundles did not expand following extraction with Triton, indicating that they coiled passively rather than due to pressure from the cell. Bundles consisted of helically wound NFs, which may provide flexibility necessary for turning of growing axons during pathfinding. Interactions between NFs and other cytoskeletal elements may be disrupted en masse during neurite retraction or regionally during remodeling. It is suggested that bundles within long axons that cannot be fully retracted into the soma could provide maintain proximal support yet still allow more distal flexibility for remodeling and changing direction during pathfinding.

Melanoma tumour vasculature heterogeneity: from mice models to human.

Tumour angiogenesis is defined by an anarchic vasculature and irregularities in alignment of endothelial cells. These structural abnormalities could explain the variability in distribution of nanomedicines in various tumour models. Then, the main goal of this study was to compare and to characterize the tumour vascular structure in different mouse models of melanoma tumours (B16F10 and SK-Mel-28) and in human melanomas from different patients. Tumours were obtained by subcutaneous injection of 106 B16F10 and 3.106 SK-Mel-28 melanoma cells in C57BL/6 and nude mice, respectively. Tumour growth was evaluated weekly, while vasculature was analysed through fluorescent labelling via CD31 and desmin. Significant differences in tumour growth and mice survival were evidenced between the two melanoma models. A fast evolution of tumours was observed for B16F10 melanoma, reaching a tumour size of 100 mm3 in 7 days compared to SK-Mel-28 which needed 21 days to reach the same volumes. Important differences in vascularization were exposed between the melanoma models, characterized by a significant enhancement of vascular density and a significant lumen size for mice melanoma models compared to human. Immunostaining revealed irregularities in endothelium structure for both melanoma models, but structural differences of vasculature were observed, characterized by a stronger expression of desmin in SK-Mel-28 tumours. While human melanoma mainly develops capillaries, structural irregularities are also observed on the samples of this tumour model. Our study revealed an impact of cell type and tumour progression on the structural vasculature of melanoma, which could impact the distribution of drugs in the tumour environment.

Immunohistochemical Method and Histopathology Judging for the Systemic Synuclein Sampling Study (S4).

Immunohistochemical (IHC) α-synuclein (Asyn) pathology in peripheral biopsies may be a biomarker of Parkinson disease (PD). The multi-center Systemic Synuclein Sampling Study (S4) is evaluating IHC Asyn pathology within skin, colon and submandibular gland biopsies from 60 PD and 20 control subjects. Asyn pathology is being evaluated by a blinded panel of specially trained neuropathologists. Preliminary work assessed 2 candidate immunoperoxidase methods using a set of PD and control autopsy-derived sections from formalin-fixed, paraffin-embedded blocks of the 3 tissues. Both methods had 100% specificity; one, utilizing the 5C12 monoclonal antibody, was more sensitive in skin (67% vs 33%), and was chosen for further use in S4. Four trainee neuropathologists were trained to perform S4 histopathology readings; in subsequent testing, their scoring was compared to that of the trainer neuropathologist on both glass slides and digital images. Specificity and sensitivity were both close to 100% with all readers in all tissue types on both glass slides and digital images except for skin, where sensitivity averaged 75% with digital images and 83.5% with glass slides. Semiquantitative (0-3) density score agreement between trainees and trainer averaged 67% for glass slides and 62% for digital images.

Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions.

OBJECTIVE: Sporadic amyotrophic lateral sclerosis (sALS) is a fatal neurodegenerative disorder affecting upper and lower motor neurons. In view of the heterogeneous presentation of the disease, one of the current challenges is to identify diagnostic and prognostic markers in order to diagnose sALS at early stage and to stratify patients in trials. In this study, we sought to identify cytological hallmarks of sALS in patient-derived fibroblasts with the aim of finding new clinical-related markers of the disease. METHODS: Primary fibroblasts were prospectively collected from patients affected with classical, rapid, and slow forms of sALS. TDP-43 localization, cytoskeleton distribution, mitochondrial network architecture, and stress granules formation were analyzed using 3D fluorescence microscopy and new super-resolution imaging. Intracellular reactive oxygen species (ROS) production was assessed using live imaging techniques. RESULTS: Six sALS patients (two classical, two rapid, and two slow) and four age-matched controls were included. No difference in fibroblasts cell growth, morphology, and distribution was noticed. The analysis of TDP-43 did not reveal any mislocalization nor aggregation of the protein. The cytoskeleton was harmoniously distributed among the cells, without any inclusion noticed, and no difference was observed regarding the mitochondrial network architecture. Basal ROS production and response to induced stress were similar among patient and control fibroblasts. CONCLUSIONS: ALS cytological lesions are absent in patient-derived fibroblasts and thus cannot contribute as diagnostic nor prognostic markers of the disease.

The accumulation of assembly intermediates of the mitochondrial complex I matrix arm is reduced by limiting glucose uptake in a neuronal-like model of MELAS syndrome.

Ketogenic diet (KD) which combined carbohydrate restriction and the addition of ketone bodies has emerged as an alternative metabolic intervention used as an anticonvulsant therapy or to treat different types of neurological or mitochondrial disorders including MELAS syndrome. MELAS syndrome is a severe mitochondrial disease mainly due to the m.3243A > G mitochondrial DNA mutation. The broad success of KD is due to multiple beneficial mechanisms with distinct effects of very low carbohydrates and ketones. To evaluate the metabolic part of carbohydrate restriction, transmitochondrial neuronal-like cybrid cells carrying the m.3243A > G mutation, shown to be associated with a severe complex I deficiency was exposed during 3 weeks to glucose restriction. Mitochondrial enzyme defects were combined with an accumulation of complex I (CI) matrix intermediates in the untreated mutant cells, leading to a drastic reduction in CI driven respiration. The severe reduction of CI was also paralleled in post-mortem brain tissue of a MELAS patient carrying high mutant load. Importantly, lowering significantly glucose concentration in cell culture improved CI assembly with a significant reduction of matrix assembly intermediates and respiration capacities were restored in a sequential manner. In addition, OXPHOS protein expression and mitochondrial DNA copy number were significantly increased in mutant cells exposed to glucose restriction. The accumulation of CI matrix intermediates appeared as a hallmark of MELAS pathophysiology highlighting a critical pathophysiological mechanism involving CI disassembly, which can be alleviated by lowering glucose fuelling and the induction of mitochondrial biogenesis, emphasizing the usefulness of metabolic interventions in MELAS syndrome.

Myoadenylate deaminase deficiency: a frequent cause of muscle pain A case detected by exercise testing.

Myoadenylate deaminase deficit (MAD, MIM#615511) is the most common cause of metabolic myopathies with an estimated prevalence of 1-2% in the general population. We report the case of a 39-year-old man suffering from severe skeletal muscle pain that had developed gradually for 4 years. A moderate increase in creatine kinase (CK) was the only biological sign observed. This study takes a closer look at a common but poorly known pathology and highlights the interest of the dynamic metabolic investigations carried out during exercise stress test with a cycle ergometer. Our non-invasive clinical and biological examination, at the interface between physiology and biology, disclosed the total absence of a physiological increase in plasma ammonia evocative of MAD. However, MAD was later confirmed by histochemistry and molecular studies, which revealed the presence of the recurrent homozygous pathogenic variant affecting the adenosine monophosphate deaminase 1 gene (AMPD1) in most patients with MAD.

Therapeutic plasma exchange in chronic dysimmune peripheral neuropathies: A 10-year retrospective study.

INTRODUCTION: Therapeutic plasma exchange (TPE) can be proposed in the treatment of chronic dysimmune peripheral neuropathies (CDPN). Actual guidelines are however based on few studies, and indications and protocols still remain to be clarified. We conducted a 10-year retrospective study in order to assess the effectiveness and tolerance of TPE in CDPN. METHODS: All patients treated for CDPN with TPE from October 2006 to March 2016 in the university hospital of Angers were included. Patients were considered responders when they presented a clinical improvement substantial enough to continue the treatment. The Hughes functional grading score was also determined for each patient before and after TPE initiation. RESULTS: Among the 206 patients who received TPE during the study period, 30 (14.6%) met the diagnostic criteria of CDPN. Four of the five paraprotein neuropathies (PPN) patients (80%) and 8 of the 11 chronic inflammatory demyelinating polyneuropathies (CIDP) patients (72.7%) were responders, with a significant improvement of the Hughes score for the latter (P = 0.013). None of the three Lewis-Sumner and the two POEMS patients showed substantial improvement. Six of the nine anti-MAG neuropathy patients (66.7%) responded to treatment, with a trend towards improvement of the Hughes score (P = 0.072). CONCLUSION: TPE appears to be effective in CIDP and PPN, and ineffective in Lewis-Sumner and POEMS syndromes. Interestingly, anti-MAG neuropathy patients showed a good rate of response to TPE. Regarding these preliminary results, a randomized trial would be very worthwhile in this disease for which there is no evidence based treatment to date.