Background-deflection Brillouin microscopy reveals altered biomechanics of intracellular stress granules by ALS protein FUS

Altered cellular biomechanics have been implicated as key photogenic triggers in age-related diseases. An aberrant liquid-to-solid phase transition, observed in in vitro reconstituted droplets of FUS protein, has been recently proposed as a possible pathogenic mechanism for amyotrophic lateral sclerosis (ALS). Whether such transition occurs in cell environments is currently unknown as a consequence of the limited measuring capability of the existing techniques, which are invasive or lack of subcellular resolution. Here we developed a non-contact and label-free imaging method, named background-deflection Brillouin microscopy, to investigate the three-dimensional intracellular biomechanics at a sub-micron resolution. Our method exploits diffraction to achieve an unprecedented 10,000-fold enhancement in the spectral contrast of single-stage spectrometers, enabling, to the best of our knowledge, the first direct biomechanical analysis on intracellular stress granules containing ALS mutant FUS protein in fixed cells. Our findings provide fundamental insights on the critical aggregation step underlying the neurodegenerative ALS disease

Protein clustering in chemically stressed HeLa cells studied by infrared nanospectroscopy

Photo-Thermal Induced Resonance (PTIR) nanospectroscopy, tuned towards amide-I absorption, was used to study the distribution of proteic material in 34 different HeLa cells, of which 18 were chemically stressed by oxidative stress with Na3AsO3. The cell nucleus was found to provide a weaker amide-I signal than the surrounding cytoplasm, while the strongest PTIR signal comes from the perinuclear region. AFM topography shows that the cells exposed to oxidative stress undergo a volume reduction with respect to the control cells, through an accumulation of the proteic material around and above the nucleus. This is confirmed by the PTIR maps of the cytoplasm, where the pixels providing a high amide-I signal were identified with a space resolution of ∼300 × 300 nm. By analyzing their distribution with two different statistical procedures we found that the probability to find protein clusters smaller than 0.6 μm in the cytoplasm of stressed HeLa cells is higher by 35% than in the control cells. These results indicate that it is possible to study proteic clustering within single cells by label-free optical nanospectroscopy

Direct conversion of human pluripotent stem cells into cranial motor neurons using a piggyBac vector

Human pluripotent stem cells (PSCs) are widely used for in vitro disease modeling. One of the challenges in the field is represented by the ability of converting human PSCs into specific disease-relevant cell types. The nervous system is composed of a wide variety of neuronal types with selective vulnerability in neurodegenerative diseases. This is particularly relevant for motor neuron diseases, in which different motor neurons populations show a different susceptibility to degeneration. Here we developed a fast and efficient method to convert human induced Pluripotent Stem Cells into cranial motor neurons of the branchiomotor and visceral motor subtype. These populations represent the motor neuron subgroup that is primarily affected by a severe form of amyotrophic lateral sclerosis with bulbar onset and worst prognosis. This goal was achieved by stable integration of an inducible vector, based on the piggyBac transposon, allowing controlled activation of Ngn2, Isl1 and Phox2a (NIP). The NIP module effectively produced electrophysiologically active cranial motor neurons. Our method can be easily extended to PSCs carrying disease-associated mutations, thus providing a useful tool to shed light on the cellular and molecular bases of selective motor neuron vulnerability in pathological conditions

Ophthalmological findings in myotonic dystrophy

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Importin-beta and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function

Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2–SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-β and CRM1 play essential roles in localising the RANBP2–SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO- conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2

FUS mutant human motoneurons display altered transcriptome and microRNA pathways with implications for ALS pathogenesis

The FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS

D-Aspartate treatment stimulates differentiation of oligodendrocyte precursors, prevents demyelination and accelerates remyelination in the cuprizone mouse model of Multiple Sclerosis

Emerging evidence support a role for some D-Aminoacids as neurotrasmitters and neuromodulators, since they are found in mammalian tissues and also in the central nervous system (CNS) (Hashimoto and Oka, 1997). They play important roles in some physiological processes, including dendritic morphology, synaptic plasticity and cognition (Wolosker et al., 2008; Billard, 2012). Among D-Aminoacids, recent studies suggest that D-Aspartic acid (D-Asp), a newly discovered agonist for NMDA receptors, play a role in NMDA receptor-dependent processes such as synaptic plasticity and memory (Errico et al., 2015). The D-Asp was described in the multi-lamellar membrane that insulate axons (Fisher et al., 1986) and its effects on the hormone biosynthesis and release have been largely explored in the years (Gold and Voskuhl 2009; Nuñez et al., 2000; Cerget et al., 2006). The exact mechanism of myelination process is still unknown, but emerging studies demonstrated the importance of intracellular changes in [Ca2+]i levels during myelination and remyelination processes (Soliven et al., 2001). Indeed, differentiation of oligodendrocyte precursors cells (OPC) and remyelination are associated with NMDARs-dependent [Ca2+]i changes (Martinez-Lozada et al., 2014). A recent work performed by our research group demonstrated that [Ca2+]i signaling mediated by the Na+/Ca2+ exchanger NCX3 plays an important role during oligodendrocytes differentiation and myelin formation (Boscia et al., 2012; Casamassa et al., 2016). In the present study, we investigated the effects of D-Asp during the OPC differentiation and remyelination by using both in vitro and in vivo techniques. In vitro, we evaluated the effects of D-Asp exposure both in human oligodendrocyte MO3.13 cell line and rat primary OPC, exposed to different concentrations of D-Aspartic acid (10-100-200 µM). Quantitative RT-PCR analyses showed that 10-200 μM D-Asp exposure for 3 days, upregulated, in a concentration-dependent manner, both the myelin markers CNPase and MBP and NCX3 transcripts in human oligodendrocytes M03.13 progenitors. The transcripts increase were significantly prevented by the NMDA receptor antagonist 10 µM MK-801 and the two NCX3 blockers, 30nM YM-244769 and 100nM BED. In accordance, microfluorimetric studies demonstrated that 100μM D-Asp administration induced an initial calcium peak of intracellular Ca2+ concentration [Ca2+]i followed by an oscillatory [Ca2+]i pattern both in oligodendrocyte MO3.13 progenitors and rat primary OPC. The NMDA antagonist 10µM MK-801 completly suppressed [Ca2+]i oscillations but only partially affected the first [Ca2+]i peak. Similar effects were observed in presence of the two selective blockers for NCX3, 30nM YM-244769 and 100nM BED. In addition, electrophysiological recordings performed in oligodendrocytes M03.13 progenitors showed that the current elicited by 100 µM D-Asp stimulation were dependent by AMPA activation, since the AMPA receptor inhibitor 10μM DNQX significantly prevented D-Asp induced inward currents. Our in vitro results suggest that D-Asp stimulates oligodendrocyte development through a mechanism involving calcium signaling through the glutamate receptors AMPA and NMDA and the Na+/Ca2+exchanger NCX3. Next, we investigated the effects of D-Asp administration in an in vivo model of demyelination/remyelination, the cuprizone mouse model. D-Asp was given during cuprizone feeding (demyelination), or after cuprizone withdrawal (remyelination). In both conditions, D-Asp treatment improved motor coordination performance in the beam balance and rotarod test. When given during demyelination D-Asp prevented MBP loss and reduced inflammation, as revealed by Western Blot analysis of MBP, Iba1 and GFAP proteins and quantitative coexpression analysis of MBP with the axonal marker NF200. Finally, electron microscopy performed on corpus callosum sections showed that D-Asp treatment accelerates remyelination in cuprizone mice, as demonstrated by the increased number in myelinated axons if compared to untreated cuprizone mice. Collectively, our results show that treatment with D-Aspartate, by influencing calcium signaling in oligodendrocytes, might produce beneficial effects during demyelination and remyelination processes

ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cells- derived motoneurons

Patient-derived induced Pluripotent Stem Cells (iPSCs) provide an opportunity to study human diseases mainly in those cases where no suitable model systems are available. Here we have taken advantage of in vitro iPSCs derived from patients affected by Amyotrophic Lateral Sclerosis and carrying mutations in the RNA-binding proteins FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patients' fibroblasts, while in the case of the severe FUS(P525L) mutation, where fibroblasts were not available, a heterozygous and a homozygous iPSC lines were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, nicely correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis

Redox-sensitive small GTPase H-Ras in murine astrocytes, an in vitro study

Although the protooncogenes small GTPases Ras are redox-sensitive proteins, how they are regulated by redox signaling in the central nervous system (CNS) is still poorly understood. Alteration in redox-sensitive targets by redox signaling may have myriad effects on Ras stability, activity and localization. Redox-mediated changes in astrocytic RAS may contribute to the control of redox homeostasis in the CNS that is connected to the pathogenesis of many diseases

3D bioprinted human cortical neural constructs derived from induced pluripotent stem cells

Bioprinting techniques use bioinks made of biocompatible non-living materials and cells to build 3D constructs in a controlled manner and with micrometric resolution. 3D bioprinted structures representative of several human tissues have been recently produced using cells derived by differentiation of induced pluripotent stem cells (iPSCs). Human iPSCs can be differentiated in a wide range of neurons and glia, providing an ideal tool for modeling the human nervous system. Here we report a neural construct generated by 3D bioprinting of cortical neurons and glial precursors derived from human iPSCs. We show that the extrusion-based printing process does not impair cell viability in the short and long term. Bioprinted cells can be further differentiated within the construct and properly express neuronal and astrocytic markers. Functional analysis of 3D bioprinted cells highlights an early stage of maturation and the establishment of early network activity behaviors. This work lays the basis for generating more complex and faithful 3D models of the human nervous systems by bioprinting neural cells derived from iPSCs