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The 3D complexity of biological tissues and the intricate structural-functional connections call for modern X-ray imaging approaches to overcome the limitations of classical imaging. Unlike other imaging techniques, X-ray phase-contrast tomography (XPCT) offers an unprecedented hierarchical 3D imaging approach to investigate different disease-relevant networks at levels ranging from the single cell through to the intact organ as a whole. We study the evolution of tissue damage and inflammation in different organs affected by the disease in the murine model for multiple sclerosis (MS), a demyelinating autoimmune disorder of the central nervous system (CNS). XPCT identifies and monitors structural and cellular alterations throughout the CNS, but also in the gut and eye, of mice induced to develop MS-like disease and sacrificed at pre-symptomatic and symptomatic time points. This study provides the sequential evolution of multi-organ damages in MS murine model showing the disease development and progression which is of obvious relevance for the human case.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

Recent studies reported that the uptake of [18F]-fluorodeoxyglucose (FDG) is increased in the spinal cord (SC) and decreased in the motor cortex (MC) of patients with ALS, suggesting that the disease might differently affect the two nervous districts with different time sequence or with different mechanisms. Here we show that MC and SC astrocytes harvested from newborn B6SJL-Tg (SOD1G93A) 1Gur mice could play different roles in the pathogenesis of the disease. Spectrophotometric and cytofluorimetric analyses showed an increase in redox stress, a decrease in antioxidant capacity and a relative mitochondria respiratory uncoupling in MC SOD1G93A astrocytes. By contrast, SC mutated cells showed a higher endurance against oxidative damage, through the increase in antioxidant defense, and a preserved respiratory function. FDG uptake reproduced the metabolic response observed in ALS patients: SOD1G93A mutation caused a selective enhancement in tracer retention only in mutated SC astrocytes, matching the activity of the reticular pentose phosphate pathway and, thus, of hexose-6P dehydrogenase. Finally, both MC and SC mutated astrocytes were characterized by an impressive ultrastructural enlargement of the endoplasmic reticulum (ER) and impairment in ER–mitochondria networking, more evident in mutated MC than in SC cells. Thus, SOD1G93A mutation differently impaired MC and SC astrocyte biology in a very early stage of life.

Background. We recently reported that enhanced [18F]-fluorodeoxyglucose (FDG) uptake in skeletal muscles predicts disease aggressiveness in patients with amyotrophic lateral sclerosis (ALS). The present experimental study aimed to assess whether this predictive potential reflects the link between FDG uptake and redox stress that has been previously reported in different tissues and disease models. Methods. The study included 15 SOD1G93A mice (as experimental ALS model) and 15 wildtype mice (around 120-days-old). Mice were submitted to micro-PET imaging. Enzymatic pathways and response to oxidative stress were evaluated in harvested quadriceps and hearts by biochemical, immunohistochemical and immunofluorescence analysis. Colocalization between the endoplasmic reticulum (ER) and the fluorescent FDG analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was performed in fresh skeletal muscle sections. Finally, mitochondrial ultrastructure and bioenergetics were evaluated in harvested quadriceps and hearts.Results. FDG retention was significantly higher in hindlimb skeletal muscles of symptomatic SOD1G93A mice with respect to control ones. This difference was not explained by any acceleration in glucose degradation through glycolysis or cytosolic pentose phosphate pathway (PPP). Similarly, it was independent of inflammatory infiltration. Rather, the high FDG retention in SOD1G93A skeletal muscle was associated with an accelerated generation of reactive oxygen species. This redox stress selectively involved the ER and the local PPP triggered by hexose-6P-dehydrogenase. ER involvement was confirmed by the colocalization of the 2-NBDG with a vital ER tracker. The oxidative damage in transgenic skeletal muscle was associated with a severe impairment in the crosstalk between ER and mitochondria combined with alterations in mitochondrial ultrastructure and fusion/fission balance. The expected respiratory damage was confirmed by a deceleration in ATP synthesis and oxygen consumption rate. These same abnormalities were represented to a markedly lower degree in the myocardium, as a sample of non-voluntary striated muscle.Conclusion. Skeletal muscle of SOD1G93A mice reproduces the increased FDG uptake observed in ALS patients. This finding reflects the selective activation of the ER-PPP in response to significant redox stress associated with alterations of mitochondrial ultrastructure, networking, and connection with the ER itself. This scenario is less severe in cardiomyocytes suggesting a relevant role for either communication with synaptic plaque or contraction dynamics.

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The degenerative effects of multiple sclerosis at the level of the vascular and neuronal networks in the central nervous system are currently the object of intensive investigation. Preclinical studies have demonstrated the efficacy of mesenchymal stem cell (MSC) therapy in experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis, but the neuropathology of specific lesions in EAE and the effects of MSC treatment are under debate. Because conventional imaging techniques entail protocols that alter the tissues, limiting the reliability of the results, we have used non-invasive X-ray phase-contrast tomography to obtain an unprecedented direct 3D characterization of EAE lesions at micro-to-nano scales, with simultaneous imaging of the vascular and neuronal networks. We reveal EAE-mediated alterations down to the capillary network. Our findings shed light on how the disease and MSC treatment affect the tissues, and promote X-ray phase-contrast tomography as a powerful tool for studying neurovascular diseases and monitoring advanced therapies.

The investigation of the neuronal network in mouse spinal cord models represents the basis for the research on neurodegenerative diseases. In this framework, the quantitative analysis of the single elements in different districts is a crucial task. However, conventional 3D imaging techniques do not have enough spatial resolution and contrast to allow for a quantitative investigation of the neuronal network. Exploiting the high coherence and the high flux of synchrotron sources, X-ray Phase-Contrast multiscale-Tomography allows for the 3D investigation of the neuronal microanatomy without any aggressive sample preparation or sectioning. We investigated healthy-mouse neuronal architecture by imaging the 3D distribution of the neuronal-network with a spatial resolution of 640 nm. The high quality of the obtained images enables a quantitative study of the neuronal structure on a subject-by-subject basis. We developed and applied a spatial statistical analysis on the motor neurons to obtain quantitative information on their 3D arrangement in the healthy-mice spinal cord. Then, we compared the obtained results with a mouse model of multiple sclerosis. Our approach paves the way to the creation of a “database” for the characterization of the neuronal network main features for a comparative investigation of neurodegenerative diseases and therapies.

Expression and function of the immunoregulatory molecule HLA-E was investigated in patients with relapsing-remitting (RR) multiple sclerosis (MS). Serum and cerebrospinal fluid (CSF) soluble (s)HLA-E and -G levels were measured by ELISA in 80 RRMS patients. Controls were patients with other inflammatory neurological disorders (OIND, n = 81) and noninflammatory neurological disorders (NIND, n = 86). Serum sHLA-E concentrations were higher in RRMS than in NIND patients only. CSF sHLA-E concentrations were higher in RRMS than controls. Increased CSF sHLA-E levels were detected in MRI inactive and clinically stable RRMS patients. sHLA-E intrathecal synthesis (ITS) was higher in RRMS than controls, and the number of patients with sHLA-E ITS above cut-off was higher i) in MS than controls, and ii) in clinically stable than clinically active MS patients. sHLA-E CSF levels and ITS correlated with i) the same sHLA-G parameters, and ii) disease duration. HLA-E expression and co-expression with CD markers were investigated in MS plaques from three different cases by immunohistochemistry and confocal microscopy, respectively. Infiltrating T lymphocytes and macrophages, as well as resident microglial cells and astrocytes expressed HLA-E. CSF samples from MS patients were finally tested for inhibitory activity of in vitro CTL and NK cell mediated cytotoxicity. sHLA-E⁺ were more effective than sHLA-E⁻ CSF samples in such inhibition. Maximum inhibition was achieved with sHLA-E⁺/sHLA-G⁺ CSF samples In conclusion, increased sHLA-E CSF levels may play an immunomodulatory role in MS, contributing to the inhibition of intrathecal inflammatory response. The potential of sHLA-E as biomarker of MS activity warrants further investigation.

In eukaryotic cells several physiologic and pathologic conditions generate the accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. To restore normal function, some ER transmembrane proteins sense the ER stress and activate coordinated signalling pathways collectively called the Unfolded Protein Response (UPR). Little is known on how the UPR relates to post-ER compartments and to the export from the ER of newly synthesized proteins. Here, we report that the ER stress response induced by either thapsigargin or nitric oxide modifies the dynamics of the intracellular distribution of ERGIC-53 and GM130, two markers of the ER Golgi Intermediate Compartment and of the cis-Golgi, respectively. In addition, induction of ER stress alters the morphology of the ERGIC and the Golgi complex and interferes with the reformation of both compartments. Moreover, ER stress rapidly reduces the transport to the Golgi complex of the temperature sensitive mutant of the Vesicular Stomatitis Virus G Glycoprotein (VSV-G) fused with the Green Fluorescent Protein (ts045G), without apparently decreasing the amount of the protein competent for export. Interestingly, a parallel rapid reduction of the number of Sec31 labelled fluorescent puncta on the ER membranes does occur, thus suggesting that the ER stress alters the ER export and the dynamic of post-ER compartments by rapidly targeting the formation of COPII-coated transport intermediates.

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific G protein-coupled receptor exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Loss of OA1 function leads to the formation of macromelanosomes, suggesting that this receptor is implicated in organelle biogenesis, however the mechanism involved in the pathogenesis of the disease remains obscure. We report here the identification of an unexpected abnormality in melanosome distribution both in retinal pigment epithelium (RPE) and skin melanocytes of Oa1-knock-out (KO) mice, consisting in a displacement of the organelles from the central cytoplasm towards the cell periphery. Despite their depletion from the microtubule (MT)-enriched perinuclear region, Oa1-KO melanosomes were able to aggregate at the centrosome upon disruption of the actin cytoskeleton or expression of a dominant-negative construct of myosin Va. Consistently, quantification of organelle transport in living cells revealed that Oa1-KO melanosomes displayed a severe reduction in MT-based motility; however, this defect was rescued to normal following inhibition of actin-dependent capture at the cell periphery. Together, these data point to a defective regulation of organelle transport in the absence of OA1 and imply that the cytoskeleton might represent a downstream effector of this receptor. Furthermore, our results enlighten a novel function for OA1 in pigment cells and suggest that ocular albinism type 1 might result from a different pathogenetic mechanism than previously thought, based on an organelle-autonomous signalling pathway implicated in the regulation of both membrane traffic and transport.

Most lysosomal storage disorders (LSDs) are caused by deficiencies of lysosomal hydrolases. While LSDs were among the first inherited diseases for which the underlying biochemical defects were identified, the mechanisms from enzyme deficiency to cell death are poorly understood. Here we show that lysosomal storage impairs autophagic delivery of bulk cytosolic contents to lysosomes. By studying the mouse models of two LSDs associated with severe neurodegeneration, multiple sulfatase deficiency (MSD) and mucopolysaccharidosis type IIIA (MPSIIIA), we observed an accumulation of autophagosomes resulting from defective autophagosome-lysosome fusion. An impairment of the autophagic pathway was demonstrated by the inefficient degradation of exogenous aggregate-prone proteins (i.e. expanded huntingtin and mutated alpha-synuclein) in cells from LSD mice. This impairment resulted in massive accumulation of polyubiquitinated proteins and of dysfunctional mitochondria which are the putative mediators of cell death. These data identify LSDs as ‘autophagy disorders’ and suggest the presence of common mechanisms in the pathogenesis of these and other neurodegenerative diseases.

Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.

X-linked recessive ocular albinism type I (OA1) is due to mutations in the OA1 gene (approved gene symbol GPR143), which is expressed in the retinal pigment epithelium (RPE). The Oa1 (Gpr143) knockout mouse (Oa1(-/-)) model recapitulates many of the OA1 retinal morphological anomalies, including a lower number of melanosomes of increased size in the RPE. The Oa1(-/-) mouse also displays some of the retinal developmental abnormalities observed in albino patients such as misrouting of the optic tracts. Here, we show that these anomalies are associated with retinal electrophysiological abnormalities, including significant decrease in a- and b-wave amplitude and delayed recovery of b-wave amplitude from photoreceptor desensitization following bright light exposure. This suggests that lack of Oa1 in the RPE impacts on photoreceptor activity. More interestingly, adeno-associated viral vector-mediated Oa1 gene transfer to the retina of the Oa1(-/-) mouse model results in significant recovery of its retinal functional abnormalities. In addition, Oa1 retinal gene transfer increases the number of melanosomes in the Oa1(-/-) mouse RPE. Our data show that gene transfer to the adult retina unexpectedly rescues both functional and morphological abnormalities in a retinal developmental disorder, opening novel potential therapeutic perspectives for this and other forms of albinism.

PURPOSE. The authors took advantage of the Oal mutant mouse in combination with other albinism mouse models (i.e., Tyrosinase and membrane-associated transporter protein [Matp]) to study the function of Oal, the gene mutated in ocular albinism type 1, in the RPE during development and after birth. METHODS. Enzyme activity and protein localization were analyzed by immunohistochemistry of tyrosinase (Tyr) in Oal-null mice. Ultrastructural analysis and morphometry were performed by electron microscopy, of the RPE in Oal-knockout mouse and double-mutant mice of Oal with either Tyr or Matp. RESULTS. Differently from other albinism models, Tyr activity was not impaired in Oa1 -/- eyes. Hypopigmentation of the RPE in Oa1 -/- mice is due to a reduced number of melanosomes. Analysis of Oa1 -/- ;Tyr c-2J /Tyr c-2J and Oa1 -/- ;Matp uw / Matp double-knockout mice, which display a block at stages II and III of melanosome maturation, respectively, revealed that Oal controls the rate of melanosome biogenesis at early stages of the organellogenesis, whereas the control on the organelle size is exerted at the final stage of melanosome development (stage IV). CONCLUSIONS. The findings indicate that Oal is involved in the regulation of melanosome maturation at two steps. Acting at early maturation stages, Oal controls the abundance of melanosomes in RPE cells. At later stages, Oal has a function in the maintenance of a correct melanosomal size. This study helps to define ocular albinism type 1 as a defect in melanosome organellogenesis and not in melanin production.

X-linked recessive Type I Ocular Albinism (OA1) is a developmental disorder of the retina due to mutations in the OA1 gene expressed in the retinal pigment epithelium (RPE). The disease is characterized by foveal hypoplasia, misrouting of the optic fibers at the chiasm and RPE ultrastructural abnormalities. The Oa1 -/- mouse recapitulates many of the OA1 retinal morphological anomalies including a lower number of melanosomes of increased size in the RPE. To test the functionality of the Oa1 -/- retina we measured by electrophysiological methods its ability to dark adapt, which is impaired in forms of albinism other than OA1. Control C57/BL6 mice showed a characteristic kinetic of recovery of the b-wave amplitude within 60 min following exposure to a pre-adapting saturating light. In contrast, the recovery of photoreceptor responses was significantly delayed in Oa1 -/- mice. To test whether Adeno-Associated-Vector-(AAV) mediated Oa1 gene transfer was able to revert this functional abnormality and the characteristic melanosome landmarks of the Oa1 phenotype, two groups of animals at different ages were injected subretinally with a mixture of AAV2/1-CMV-Oa1+AAV2/1-EGFP in the right eye and the same amount of AAV2/1-CMV-EGFP in the left eye. Dark adaptation in both eyes and groups was analyzed 1 month after gene transfer. The Oa1 -/- eyes that received only EGFP showed a delayed ability to dark adapt similar to the eyes of untreated Oa1 mutant mice. In the eyes that received the therapeutic vector the recovery was complete in 60 min: as in wild type animals. Electron microscopy analysis revealed a significant increase in the number of melanosomes in the RPE of the retina treated precociously with AAV. Our results suggest that post-natal Oa1 gene transfer in mice rescues some of the functional and morphological abnormalities that develop in the Oa1 -/- retina.