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Dermic fibroblasts have been proposed as a potential genetic-ALS cellular model. This study aimed to explore whether dermic fibroblasts from patients with sporadic-ALS (sALS) recapitulate alterations typical of ALS motor neurons and exhibit abnormal DNA-damage response. Dermic fibroblasts were obtained from eight sALS patients and four control subjects. Cellular characterization included proliferation rate analysis, cytoarchitecture studies and confocal immunofluorescence assessment for TDP-43. Additionally, basal and irradiation-induced DNA damage was evaluated by confocal immunofluorescence and biochemical techniques. sALS-fibroblasts showed decreased proliferation rates compared to controls. Additionally, whereas control fibroblasts exhibited the expected normal spindle-shaped morphology, ALS fibroblasts were thinner, with reduced cell size and enlarged nucleoli, with frequent cytoplasmic TDP-43aggregates. Also, baseline signs of DNA damage were evidenced more frequently in ALS-derived fibroblasts (11 versus 4% in control-fibroblasts). Assays for evaluating the irradiation-induced DNA damage demonstrated that DNA repair was defective in ALS-fibroblasts, accumulating more than double of γH2AX-positive DNA damage foci than controls. Very intriguingly, the proportion of fibroblasts particularly vulnerable to irradiation (with more than 15 DNA damage foci per nucleus) was seven times higher in ALS-derived fibroblasts than in controls. Dermic-derived ALS fibroblasts recapitulate relevant cellular features of sALS and show a higher susceptibility to DNA damage and defective DNA repair responses. Altogether, these results support that dermic fibroblasts may represent a convenient and accessible ALS cellular model to study pathogenetic mechanisms, particularly those related to DNA damage response, as well as the eventual response to disease-modifying therapies.

The Purkinje cell (PC) degeneration (pcd) mouse harbors a mutation in Agtpbp1 gene that encodes for the cytosolic carboxypeptidase, CCP1. The mutation causes degeneration and death of PCs during the postnatal life, resulting in clinical and pathological manifestation of cerebellar ataxia. Monogenic biallelic damaging variants in the Agtpbp1 gene cause infantile-onset neurodegeneration and cerebellar atrophy, linking loss of functional CCP1 with human neurodegeneration. Although CCP1 plays a key role in the regulation of tubulin stabilization, its loss of function in PCs leads to a severe nuclear phenotype with heterochromatinization and accumulation of DNA damage. Therefore, the pcd mice provides a useful neuronal model to investigate nuclear mechanisms involved in neurodegeneration, particularly the nucleolar stress. In this study, we demonstrated that the Agtpbp1 gene mutation induces a p53-dependent nucleolar stress response in PCs, which is characterized by nucleolar fragmentation, nucleoplasmic and cytoplasmic mislocalization of nucleolin, and dysfunction of both pre-rRNA processing and mRNA translation. RT-qPCR analysis revealed reduction of mature 18S rRNA, with a parallel increase of its intermediate 18S-5’-ETS precursor, that correlates with a reduced expression of Fbl mRNA, which encodes an essential factor for rRNA processing. Moreover, nucleolar alterations were accompanied by a reduction of PTEN mRNA and protein levels, which appears to be related to the chromosome instability and accumulation of DNA damage in degenerating PCs. Our results highlight the essential contribution of nucleolar stress to PC degeneration and also underscore the nucleoplasmic mislocalization of nucleolin as a potential indicator of neurodegenerative processes., This work was supported by the following grants: “Instituto de Salud Carlos III” (CIBERNED, CB06/05/0037) and CIBERONC (CB16/12/00352), “Instituto de Investigación Valdecilla” (IDIVAL, Santander, Spain), FIS PI16/02137 from ISCIII and SAF2016-79668-R (MINECO, Spain), SA043U16 (UIC076) and SA030P17 (UIC217) from JCyL (Spain).

Type I spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by the loss or mutation of the survival motor neuron 1 (SMN1) gene. The reduction in SMN protein levels in SMA leads to the degeneration of motor neurons and muscular atrophy. In this study, we analyzed the nuclear reorganization in human skeletal myofibers from a type I SMA patient carrying a deletion of exons 7 and 8 in the SMN1 gene and two SMN2 gene copies and showing reduced SMN protein levels in the muscle compared with those in control samples. The morphometric analysis of myofiber size revealed the coexistence of atrophic and hypertrophic myofibers in SMA samples. Compared with controls, both nuclear size and the nuclear shape factor were significantly reduced in SMA myonuclei. Nuclear reorganization in SMA myonuclei was characterized by extensive heterochromatinization, the aggregation of splicing factors in large interchromatin granule clusters, and nucleolar alterations with the accumulation of the granular component and a loss of fibrillar center/dense fibrillar component units. These nuclear alterations reflect a severe perturbation of global pre-mRNA transcription and splicing, as well as nucleolar dysfunction, in SMA myofibers. Moreover, the finding of similar nuclear reorganization in both atrophic and hypetrophic myofibers provides additional support that the SMN deficiency in SMA patients may primarily affect the skeletal myofibers.

Down syndrome (DS) or trisomy of chromosome 21 (Hsa21) is characterized by impaired hippocampal-dependent learning and memory. These alterations are due to defective neurogenesis and to neuromorphological and functional anomalies of numerous neuronal populations, including hippocampal granular cells (GCs). It has been proposed that the additional gene dose in trisomic cells induces modifications in nuclear compartments and on the chromatin landscape, which could contribute to some DS phenotypes. The Ts65Dn (TS) mouse model of DS carries a triplication of 92 genes orthologous to those found in Hsa21, and shares many phenotypes with DS individuals, including cognitive and neuromorphological alterations. Considering its essential role in hippocampal memory formation, we investigated whether the triplication of this set of Hsa21 orthologous genes in TS mice modifies the nuclear architecture of their GCs. Our results show that the TS mouse presents alterations in the nuclear architecture of its GCs, affecting nuclear compartments involved in transcription and pre-rRNA and pre-mRNA processing. In particular, the GCs of the TS mouse show alterations in the nucleolar fusion pattern and the molecular assembly of Cajal bodies (CBs). Furthermore, hippocampal GCs of TS mice present an epigenetic dysregulation of chromatin that results in an increased heterochromatinization and reduced global transcriptional activity. These nuclear alterations could play an important role in the neuromorphological and/or functional alterations of the hippocampal GCs implicated in the cognitive dysfunction characteristic of TS mice.

© 2021 by the authors., Recent reports have identified rare, biallelic damaging variants of the AGTPBP1 gene that cause a novel and documented human disease known as childhood-onset neurodegeneration with cerebellar atrophy (CONDCA), linking loss of function of the AGTPBP1 protein to human neurodegenerative diseases. CONDCA patients exhibit progressive cognitive decline, ataxia, hypotonia or muscle weakness among other clinical features that may be fatal. Loss of AGTPBP1 in humans recapitulates the neurodegenerative course reported in a well-characterised murine animal model harbouring loss-of-function mutations in the AGTPBP1 gene. In particular, in the Purkinje cell degeneration (pcd) mouse model, mutations in AGTPBP1 lead to early cerebellar ataxia, which correlates with the massive loss of cerebellar Purkinje cells. In addition, neurodegeneration in the olfactory bulb, retina, thalamus and spinal cord were also reported. In addition to neurodegeneration, pcd mice show behavioural deficits such as cognitive decline. Here, we provide an overview of what is currently known about the structure and functional role of AGTPBP1 and discuss the various alterations in AGTPBP1 that cause neurodegeneration in the pcd mutant mouse and humans with CONDCA. The sequence of neuropathological events that occur in pcd mice and the mechanisms governing these neurodegenerative processes are also reported. Finally, we describe the therapeutic strategies that were applied in pcd mice and focus on the potential usefulness of pcd mice as a promising model for the development of new therapeutic strategies for clinical trials in humans, which may offer potential beneficial options for patients with AGTPBP1 mutation-related CONDCA., This work was supported by funding received by the Institute of Health Carlos III (CIBERONC, CB16/12/00352 and CIBERNED, CB/05/0037), the Valdecilla Research Institute (IDIVAL, Santander Spain), the Regional Government of Castile and Leon, funds received by the European Regional Development Fund (SA264P18-UIC076; University of Salamanca, Spain) and the Samuel-Solórzano Barruso Foundation (FS/32-2020, University of Salamanca, Spain)

There is a huge interest in developing novel hollow fiber (HF) membranes able to modulate neural differentiation to produce in vitro blood-brain barrier (BBB) models for biomedical and pharmaceutical research, due to the low cell-inductive properties of the polymer HFs used in current BBB models. In this work, poly(ε-caprolactone) (PCL) and composite PCL/graphene (PCL/G) HF membranes were prepared by phase inversion and were characterized in terms of mechanical, electrical, morphological, chemical, and mass transport properties. The presence of graphene in PCL/G membranes enlarged the pore size and the water flux and presented significantly higher electrical conductivity than PCL HFs. Biocompatibility assay showed that PCL/G HFs significantly increased C6 cells adhesion and differentiation towards astrocytes, may be attributed to their higher electrical conductivity in comparison to PCL HFs. On the other hand, PCL/G membranes produced a cytotoxic effect on the endothelial cell line HUVEC presumably related with a higher production of intracellular reactive oxygen species induced by the nanomaterial in this particular cell line. These results prove the potential of PCL HF membranes to grow endothelial cells and PCL/G HF membranes to differentiate astrocytes, the two characteristic cell types that could develop in vitro BBB models in future 3D co-culture systems.

Spinal muscular atrophy (SMA) is caused by a homozygous deletion or mutation in the survival motor neuron 1 (SMN1) gene that leads to reduced levels of SMN protein resulting in degeneration of motor neurons (MNs). The best known functions of SMN is the biogenesis of spliceosomal snRNPs. Linked to this function, Cajal bodies (CBs) are involved in the assembly of spliceosomal (snRNPs) and nucleolar (snoRNPs) ribonucleoproteins required for pre-mRNA and pre-rRNA processing. Recent studies support that the interaction between CBs and nucleoli, which are especially prominent in neurons, is essential for the nucleolar rRNA homeostasis. We use the SMN∆7 murine model of type I SMA to investigate the cellular basis of the dysfunction of RNA metabolism in MNs. SMN deficiency in postnatal MNs produces a depletion of functional CBs and relocalization of coilin, which is a scaffold protein of CBs, in snRNP-free perinucleolar caps or within the nucleolus. Disruption of CBs is the earliest nuclear sign of MN degeneration. We demonstrate that depletion of CBs, with loss of CB-nucleolus interactions, induces a progressive nucleolar dysfunction in ribosome biogenesis. It includes reorganization and loss of nucleolar transcription units, segregation of dense fibrillar and granular components, retention of SUMO-conjugated proteins in intranucleolar bodies and a reactive, compensatory, up-regulation of mature 18S rRNA and genes encoding key nucleolar proteins, such as upstream binding factor, fibrillarin, nucleolin and nucleophosmin. We propose that CB depletion and nucleolar alterations are essential components of the dysfunction of RNA metabolism in SMA. This work was supported by the following grants: “Dirección General de Investigación” of Spain (BFU2014-54754-P), “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; CB06/05/0037)” from Spain, and “Ministerio de Economía y Competitividad” of Spain cofinanced by FEDER (SAF2015-70801-R). Dr. Tapia is a recipient of a grant from SMA Europe cofinanced by FundAME (Spain) and "Instituto de Investigación Valdecilla" (IDIVAL; Next-Val) from Santander, Spain.

Spinal muscular atrophy (SMA) is caused by a deletion or mutation of the survival motor neuron 1 (SMN1) gene. Reduced SMN levels lead to motor neuron degeneration and muscular atrophy. SMN protein localizes to the cytoplasm and Cajal bodies. Moreover, in myofibrils from Drosophila and mice, SMN is a sarcomeric protein localized to the Z-disc. Although SMN participates in multiple functions, including the biogenesis of spliceosomal small nuclear ribonucleoproteins, its role in the sarcomere is unclear. Here, we analyzed the sarcomeric organization of SMN in human control and type I SMA skeletal myofibers. In control sarcomeres, we demonstrate that human SMN is localized to the titin-positive M-band and actin-positive I-band, and to SMN-positive granules that flanked the Z-discs. Co-immunoprecipitation assays revealed that SMN interacts with the sarcomeric protein actin, α-actinin, titin, and profilin2. In the type I SMA muscle, SMN levels were reduced, and atrophic (denervated) and hypertrophic (nondenervated) myofibers coexisted. The hypertrophied myofibers, which are potential primary targets of SMN deficiency, exhibited sites of focal or segmental alterations of the actin cytoskeleton, where the SMN immunostaining pattern was altered. Moreover, SMN was relocalized to the Z-disc in overcontracted minisarcomeres from hypertrophic myofibers. We propose that SMN could have an integrating role in the molecular components of the sarcomere. Consequently, low SMN levels might impact the normal sarcomeric architecture, resulting in the disruption of myofibrils found in SMA muscle. This primary effect might be independent of the neurogenic myopathy produced by denervation and contribute to pathophysiology of the SMA myopathy.

Guillain–Barre syndrome (GBS) is an acute-onset, immune-mediated disorder of the peripheral nervous system. In early GBS, arbitrarily established up to 10 days of disease onset, patients could exhibit selective manifestations due to involvement of the proximal nerves, including nerve roots, spinal nerves and plexuses. Such manifestations are proximal weakness, inaugural nerve trunk pain, and atypical electrophysiological patterns, which may lead to delayed diagnosis. The aim of this paper was to analyze the nosology of early GBS reviewing electrophysiological, autopsy and imaging studies, both in acute inflammatory demyelinating polyneuropathy (AIDP) and acute motor/motor-sensory axonal neuropathy (AMAN/AMSAN). Early electrophysiology showed either well-defined demyelinating or axonal patterns, or a non-diagnostic pattern with abnormal late responses; there may be attenuated M responses upon lumbar root stimulation as the only finding. Pathological changes predominated in proximal nerves, in some studies, most prominent at the sides where the spinal roots unite to form the spinal nerves; on very early GBS endoneurial inflammatory edema was the outstanding feature. In the far majority of cases, spinal magnetic resonance imaging showed contrast enhancement of cauda equina, selectively involving anterior roots in AMAN. Both in AIDP and AMAN/AMSAN, ultrasonography has demonstrated frequent enlargement of ventral rami of C5–C7 nerves with blurred boundaries, whereas sonograms of upper and lower extremity peripheral nerves exhibited variable and less frequent abnormalities. We provide new insights into the pathogenesis and classification of early GBS.

Cajal is commonly regarded as the father of modern neuroscience in recognition of his fundamental work on the structure of the nervous system. But Cajal also made seminal contributions to the knowledge of nuclear structure in the early 1900s, including the discovery of the “accessory body” later renamed “Cajal body” (CB). This important nuclear structure has emerged as a center for the assembly of ribonucleoproteins (RNPs) required for splicing, ribosome biogenesis and telomere maintenance. The modern era of CB research started in the 1990s with the discovery of coilin, now known as a scaffold protein of CBs, and specific probes for small nuclear RNAs (snRNAs). In this review, we summarize what we have learned in the recent decades concerning CBs in post-mitotic neurons, thereby ruling out dynamic changes in CB functions during the cell cycle. We show that CBs are particularly prominent in neurons, where they frequently associate with the nucleolus. Neuronal CBs are transcription-dependent nuclear organelles. Indeed, their number dynamically accommodates to support the high neuronal demand for splicing and ribosome biogenesis required for sustaining metabolic and bioelectrical activity. Mature neurons have canonical CBs enriched in coilin, survival motor neuron protein and snRNPs. Disruption and loss of neuronal CBs associate with severe neuronal dysfunctions in several neurological disorders such as motor neuron diseases. In particular, CB depletion in motor neurons seems to reflect a perturbation of transcription and splicing in spinal muscular atrophy, the most common genetic cause of infant mortality.

MXD1 is a protein that interacts with MAX, to form a repressive transcription factor. MXD1-MAX binds E-boxes. MXD1-MAX antagonizes the transcriptional activity of the MYC oncoprotein in most models. It has been reported that MYC overexpression leads to augmented RNA synthesis and ribosome biogenesis, which is a relevant activity in MYC-mediated tumorigenesis. Here we describe that MXD1, but not MYC or MNT, localizes to the nucleolus in a wide array of cell lines derived from different tissues (carcinoma, leukemia) as well as in embryonic stem cells. MXD1 also localizes in the nucleolus of primary tissue cells as neurons and Sertoli cells. The nucleolar localization of MXD1 was confirmed by co-localization with UBF. Co-immunoprecipitation experiments showed that MXD1 interacted with UBF and proximity ligase assays revealed that this interaction takes place in the nucleolus. Furthermore, chromatin immunoprecipitation assays showed that MXD1 was bound in the transcribed rDNA chromatin, where it co-localizes with UBF, but also in the ribosomal intergenic regions. The MXD1 involvement in rRNA synthesis was also suggested by the nucleolar segregation upon rRNA synthesis inhibition by actinomycin D. Silencing of MXD1 with siRNAs resulted in increased synthesis of pre-rRNA while enforced MXD1 expression reduces it. The results suggest a new role for MXD1, which is the control of ribosome biogenesis. This new MXD1 function would be important to curb MYC activity in tumor cells., The work was supported by grants SAF2014-53526 (to JL), BFU2014-54754P (to ML and MTB) from Spanish Economy and Competitiveness Ministry (MINECO), and grants RETIC-RD012-036-033 (to JL) and CIBERNED CB06/05/0037 (to ML) from Instituto Carlos III to JL. These funding were co-sponsored by the FEDER program. MCL was recipient of a fellowship from the FPU program from MINECO.

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2 Œ-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

There is a huge interest in developing novel hollow fiber (HF) membranes able to modulate neural differentiation to produce in vitro blood&ndash, brain barrier (BBB) models for biomedical and pharmaceutical research, due to the low cell-inductive properties of the polymer HFs used in current BBB models. In this work, poly(&epsilon, caprolactone) (PCL) and composite PCL/graphene (PCL/G) HF membranes were prepared by phase inversion and were characterized in terms of mechanical, electrical, morphological, chemical, and mass transport properties. The presence of graphene in PCL/G membranes enlarged the pore size and the water flux and presented significantly higher electrical conductivity than PCL HFs. A biocompatibility assay showed that PCL/G HFs significantly increased C6 cells adhesion and differentiation towards astrocytes, which may be attributed to their higher electrical conductivity in comparison to PCL HFs. On the other hand, PCL/G membranes produced a cytotoxic effect on the endothelial cell line HUVEC presumably related with a higher production of intracellular reactive oxygen species induced by the nanomaterial in this particular cell line. These results prove the potential of PCL HF membranes to grow endothelial cells and PCL/G HF membranes to differentiate astrocytes, the two characteristic cell types that could develop in vitro BBB models in future 3D co-culture systems.

Spinal muscular atrophy (SMA) is a severe motor neuron (MN) disease caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene, which results in reduced levels of the SMN protein and the selective degeneration of lower MNs. The best-known function of SMN is the biogenesis of spliceosomal snRNPs, the major components of the pre-mRNA splicing machinery. Therefore, SMN deficiency in SMA leads to widespread splicing abnormalities. We used the SMN∆7 mouse model of SMA to investigate the cellular reorganization of polyadenylated mRNAs associated with the splicing dysfunction in MNs. We demonstrate that SMN deficiency induced the abnormal nuclear accumulation in euchromatin domains of poly(A) RNA granules (PARGs) enriched in the splicing regulator Sam68. However, these granules lacked other RNA-binding proteins, such as TDP43, PABPN1, hnRNPA12B, REF and Y14, which are essential for mRNA processing and nuclear export. These effects were accompanied by changes in the alternative splicing of the Sam68-dependent Bcl-x and Nrnx1 genes, as well as changes in the relative accumulation of the intron-containing Chat, Chodl, Myh9 and Myh14 mRNAs, which are all important for MN functions. PARG-containing MNs were observed at presymptomatic SMA stage, increasing their number during the symptomatic stage. Moreover, the massive accumulations of poly(A) RNA granules in MNs was accompanied by the cytoplasmic depletion of polyadenylated mRNAs for their translation. We suggest that the SMN-dependent abnormal accumulation of polyadenylated mRNAs and Sam68 in PARGs reflects a severe dysfunction of both mRNA processing and translation, which could contribute to SMA pathogenesis. This work was supported by grants from: “Dirección General de Investigación” of Spain (BFU2014-54754-P and SAF2015-70801-R, cofinanced by FEDER) and “Instituto de Investigación Marqués de Valdecilla-IDIVAL (NVAL17/22). Dr. Tapia is the recipient of a grant from SMA Europe and FundAME (Spain).

Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disease affecting motor neurons (MN). This fatal disease is characterized by progressive muscular atrophy and unfortunately it does not have an effective treatment. Although a small proportion of ALS cases have a familiar origin, the vast majority of them are thought to have a sporadic origin. Although the pathogenesis of ALS has not been fully elucidated, various disorders in different cellular functions such as gene expression, protein metabolism, axonal transport and glial cell disorders have been linked to MN degeneration. Among them, proteostasis is one of the best studied. Retinoids are vitamin A-derived substances that play a crucial role in embryogenesis, development, programmed cell death and other cellular functions. Retinoid agonists behave as transcription factors throughout the activation of the nuclear retinoid receptors. Several reports in the literature suggest that retinoids are involved in proteostasis regulation, by modulating its two major pathways, the ubiquitin-proteasome system and the autophagy-lysosome response. Additionally, there are some evidences for a role of retinoids themselves, in ALS pathogenesis. In this review, we discuss the importance of proteostasis disruption as a trigger for MN degeneration and the capability of retinoids to modulate it, as well as the potential therapeutic role of retinoids as a new therapy in ALS.

Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4 Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5'-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for γH2AX and 53BP1, were observed as early as 30 min post-IR; their number progressively reduced at 3 h, 1 day, and 3 days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.

Objective Although prevailing spinal nerve involvement has been recognized in a few detailed Guillain–Barre syndrome (GBS) autopsy reports, imaging studies addressing this question in cervical nerves are lacking. Methods We describe clinical, electrophysiological, ultrasonographic (US) and pathological findings in six consecutive early GBS patients, evaluated within 10 days of onset. Results Patients’ ages ranged from 37 to 80 years. Five patients required mechanical ventilation, two of them having died 9 and 28 days after onset. Upper- and lower-limb nerve US showed abnormal findings in just 8.8% of scanned peripheral nerves. In comparison with 46 aged-matched control subjects, US of the fifth to seventh cervical nerves showed changes in four cases, which consisted of significant nerve enlargement, blurred boundaries of the corresponding ventral rami, or both. Autopsy study in one case demonstrated that pathology, consisting of demyelination and endoneurial inflammatory oedema, mainly involved cervical and lumbar nerves. Conclusions In early GBS inflammatory oedema of spinal nerves is a pathogenically relevant feature to understanding the mechanism of ascending paralysis, particularly when conventional electrophysiological studies are normal or not diagnostic. Significance Findings advocate the use of cervical nerve US in early GBS.

Neurons are highly vulnerable to DNA damage induced by genotoxic agents such as topoisomerase activity, oxidative stress, ionizing radiation (IR) and chemotherapeutic drugs. To avert the detrimental effects of DNA lesions in genome stability, transcription and apoptosis, neurons activate robust DNA repair mechanisms. However, defective DNA repair with accumulation of unrepaired DNA are at the basis of brain ageing and several neurodegenerative diseases. Understanding the mechanisms by which neurons tolerate DNA damage accumulation as well as defining the genomic regions that are more vulnerable to DNA damage or refractory to DNA repair and therefore constitute potential targets in neurodegenerative diseases are essential issues in the field. In this work we investigated the nuclear topography and organization together with the genome-wide distribution of unrepaired DNA in rat cortical neurons 15 days upon IR. About 5% of non-irradiated and 55% of irradiated cells accumulate unrepaired DNA within persistent DNA damage foci (PDDF) of chromatin. These PDDF are featured by persistent activation of DNA damage/repair signaling, lack of transcription and localization in repressive nuclear microenvironments. Interestingly, the chromatin insulator CTCF is concentrated at the PDDF boundaries, likely contributing to isolate unrepaired DNA from intact transcriptionally active chromatin. By confining damaged DNA, PDDF would help preserving genomic integrity and preventing the production of aberrant proteins encoded by damaged genes.ChIP-seq analysis of genome-wide γH2AX distribution revealed a number of genomic regions enriched in γH2AX signal in IR-treated cortical neurons. Some of these regions are in close proximity to genes encoding essential proteins for neuronal functions and human neurodegenerative disorders such as epm2a (Lafora disease), serpini1 (familial encephalopathy with neuroserpin inclusion bodies) and il1rpl1 (mental retardation, X-linked 21). Persistent γH2AX signal close to those regions suggests that nearby genes could be either more vulnerable to DNA damage or more refractory to DNA repair. Authors wish to thank Raquel García Ceballos and Daniel Giménez for technical assistance. This work was supported by the following grants: “Dirección General de Investigación”(BFU2014–54754-P) and“Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas”(CIBERNED; CB06/05/0037)Spain. Sí

Down syndrome (DS) is characterized by a marked reduction in the size of the brain and cerebellum. These changes play an important role in the motor alterations and cognitive disabilities observed in this condition. The Ts65Dn (TS) mouse, the most commonly used model of DS, reflects many DS phenotypes, including alterations in cerebellar morphology. One of the genes that is overexpressed in both individuals with DS and TS mice is DYRK1A/Dyrk1A (dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A), which has been implicated in the altered cerebellar structural and functional phenotypes observed in both populations. The aim of this study was to evaluate the effect of Dyrk1A on different alterations observed in the cerebellum of TS animals. TS mice were crossed with Dyrk1A +/- KO mice to obtain mice with a triplicate segment of Mmu16 that included Dyrk1A (TS +/+/+), mice with triplicate copies of the same genes that carried only two copies of Dyrk1A (TS +/+/-), euploid mice that expressed a normal dose of Dyrk1A (CO +/+) and CO animals with a single copy of Dyrk1A (CO +/-). Male mice were used for all experiments. The normalization of the Dyrk1A gene dosage did not rescue the reduced cerebellar volume. However, it increased the size of the granular and molecular layers, the densities of granular and Purkinje cells, and dendritic arborization. Furthermore, it improved the excitatory/inhibitory balance and walking pattern of TS +/+/- mice. These results support the hypothesis that Dyrk1A is involved in some of the structural and functional cerebellar phenotypes observed in the TS mouse model. This work was supported by grants from the Jerome Lejeune Foundation and Fundación Tatiana Pérez de Guzmán el Bueno and the Spanish Ministry of Economy and Competitiveness (PSI-2016-76194-R, AEI/FEDER, EU) and “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0037)” from Spain.

The survival of motor neuron (SMN) protein plays an essential role in the biogenesis of spliceosomal snRNPs and the molecular assembly of Cajal bodies (CBs). Deletion of or mutations in the SMN1 gene cause spinal muscular atrophy (SMA) with degeneration and loss of motor neurons. Reduced SMN levels in SMA lead to deficient snRNP biogenesis with consequent splicing pathology. Here, we demonstrate that SMN is a novel and specific target of the acetyltransferase CBP (CREB-binding protein). Furthermore, we identify lysine (K) 119 as the main acetylation site in SMN. Importantly, SMN acetylation enhances its cytoplasmic localization, causes depletion of CBs, and reduces the accumulation of snRNPs in nuclear speckles. In contrast, the acetylation-deficient SMNK119R mutant promotes formation of CBs and a novel category of promyelocytic leukemia (PML) bodies enriched in this protein. Acetylation increases the half-life of SMN protein, reduces its cytoplasmic diffusion rate and modifies its interactome. Hence, SMN acetylation leads to its dysfunction, which explains the ineffectiveness of HDAC (histone deacetylases) inhibitors in SMA therapy despite their potential to increase SMN levels.

Dendritic spines are specialised membrane protrusions of neuronal dendrites that receive the majority of excitatory synaptic inputs. Abnormal changes in their density, size and morphology have been associated with various neurological and psychiatric disorders, including those deriving from drug addiction. Dendritic spine formation, morphology and synaptic functions are governed by the actin cytoskeleton. Previous in vivo studies have shown that ethanol alters the number and morphology of spines, although the mechanisms underlying these alterations remain unknown. It has also been described how chronic ethanol exposure affects the levels, assembly and cellular organisation of the actin cytoskeleton in hippocampal neurons in primary culture. Therefore, we hypothesised that the ethanol-induced alterations in the number and shape of dendritic spines are due to alterations in the mechanisms regulating actin cytoskeleton integrity. The results presented herein show that chronic exposure to moderate levels of alcohol (30 mM) during the first 2 weeks of culture reduces dendritic spine density and alters the proportion of the different morphologies of these structures in hippocampal neurons, which affects the formation of mature spines. Apparently, these effects are associated with an increase in the G-actin/F-actin ratio due to a reduction of the F-actin fraction, leading to changes in the levels of the different factors regulating the organisation of this cytoskeletal component. The data presented herein indicate that these effects occur between weeks 1 and 2 of culture, an important period in dendritic spines development. These changes may be related to the dysfunction in the memory and learning processes present in children prenatally exposed to ethanol.

Purkinje Cell Degeneration (PCD) mice harbor a nna1 gene mutation which leads to an early and rapid degeneration of Purkinje cells (PC) between the third and fourth week of age. This mutation also underlies the death of mitral cells (MC) in the olfactory bulb (OB), but this process is slower and longer than in PC. No clear interpretations supporting the marked differences in these neurodegenerative processes exist. Growing evidence suggests that either beneficial or detrimental effects of gliosis in damaged regions would underlie these divergences. Here, we examined the gliosis occurring during PC and MC death in the PCD mouse. Our results demonstrated different glial reactions in both affected regions. PC disappearance stimulated a severe gliosis characterized by strong morphological changes, enhanced glial proliferation, as well as the release of pro-inflammatory mediators. By contrast, MC degeneration seems to promote a more attenuated glial response in the PCD OB compared with that of the cerebellum. Strikingly, cerebellar oligodendrocytes died by apoptosis in the PCD, whereas bulbar ones were not affected. Interestingly, the level of nna1 mRNA under normal conditions was higher in the cerebellum than in the OB, probably related to a faster neurodegeneration and stronger glial reaction in its absence. The glial responses may thus influence the neurodegenerative course in the cerebellum and OB of the mutant mouse brain, providing harmful and beneficial microenvironments, respectively., Grant sponsor: ‘‘Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) Instituto de Salud Carlos III’’; Grant number: CB06/05/0037; Grant sponsor: Ministerio de Ciencia y Tecnología; Grant numbers: BFU2010-18284, BFU2011-23983; Grant sponsors: ‘‘Instituto de Formación e Investigación Marqués de Valdecilla’’ (IFIMAV), Junta de Castilla y León, Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León y Fundación Memoria D. Samuel Solórzano-Barruso, Spain.

The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.

Coilin, a molecular marker for Cajal bodies (CBs), is a phosphoprotein that contains a cryptic nucleolar localization signal and multiple interacting domains, such as the RG-box. Post-translational symmetrical dimethylation of arginines on the coilin RG-box is required for the recruitment of the survival motor neuron (SMN) protein and splicing small ribonucleoproteins (snRNPs) to CBs. Here, we analyze the role of the methylation state of coilin in the regulation of its localization to the nucleolus. We use the MCF7 MTAP(-/-) cell line, which lacks the gene encoding 5'-methylthioadenosine phosphorylase (MTAP). This is a key enzyme of the methionine salvage pathway. The reduction of the levels of coilin methylation causes disruption of the canonical CBs and coilin redistribution to nucleoplasmic microfoci and to the nucleolus. Intranucleolar coilin is unmethylated and appears restricted to the dense fibrillar component. Interestingly, intranucleolar coilin is not associated with SMN or snRNPs, and does not interfere with global transcriptional activity. Overexpression of wild-type MTAP reverts the intranucleolar localization of coilin and the disruption of CBs to the normal coilin phenotype. Our results suggest the existence of a dynamic flux of coilin between CBs, nucleoplasm and nucleolus, and indicate that coilin methylation plays a key role in this process.

TDP-43 is a RNA/DNA-binding protein structurally related to nuclear hnRNP proteins. Previous biochemical studies have shown that this nuclear protein plays a role in the regulation of gene transcription, alternative splicing and mRNA stability. Despite the ubiquitous distribution of TDP-43, the growing list of TDP-43 proteinopathies is primarily associated with neurodegenerative disorders. Particularly, TDP-43 redistributes to the cytoplasm and forms pathological inclusions in amyotrophic lateral sclerosis and several forms of sporadic and familiar frontotemporal lobar degeneration. Here, we have studied the nuclear compartmentalization of TDP-43 in normal rat neurons by using light and electron microscopy immunocytochemistry with molecular markers for nuclear compartments, a transcription assay with 5'-fluorouridine, and in situ hybridization for telomeric DNA. TDP-43 is concentrated in euchromatin domains, specifically in perichromatin fibrils, nuclear sites of transcription and cotranscriptional splicing. In these structures, TDP-43 colocalizes with 5'-fluorouridine incorporation sites into nascent pre-mRNA. TDP-43 is absent in transcriptionally silent centromeric and telomeric heterochromatin, as well as in the Cajal body, a transcription free nuclear compartment. Furthermore, a weak TDP-43 immunolabeling is found in nuclear speckles of splicing factors. The specific localization of TDP-43 in active sites of transcription and cotranscriptional splicing is consistent with biochemical data indicating a role of TDP-43 in the regulation of transcription and alternative splicing.

The ubiquitin-dependent proteasome system (UPS) is the major pathway responsible for selective nuclear and cytoplasmic protein degradation. Bortezomib, a boronic acid dipeptide, is a reversible 20S proteasome inhibitor used as novel anticancer drug, particularly in the treatment of multiple myeloma and certain lymphomas. Bortezomib-induced peripheral neuropathy (BIPN) is a widely recognized dose-limiting neurotoxicity of this proteasome inhibitor, which causes a significant negative impact on the quality of life. The pathogenic mechanisms underlying bortezomib neurotoxicity are little known. In this study a rat was used as our animal model to investigate the bortezomib-induced nuclear changes in dorsal root ganglia (DRG) neurons. Our results indicate that this neuronal population is an important target of bortezomib neurotoxicity. Nuclear changes include accumulation of ubiquitin-protein conjugates, reduction of transcriptional activity, and nuclear retention of poly(A) RNAs in numerous spherical or ring-shaped dense granules. They also contained the RNA-binding proteins PABPN1 (poly(A) binding protein nuclear 1) and Sam68, but lacked the mRNA nuclear export factors REF and Y14. At the cytoplasmic level, most neurons exhibited chromatolysis, supporting the inhibition of mRNA translation. Our results indicate that bortezomib interferes with transcription, nuclear processing and transport, and cytoplasmic translation of mRNAs in DRG neurons. They also support that this neuronal dysfunction is an essential pathogenic mechanism in the BIPN, which is characterized by sensory impairment including sensory ataxia.

Nuclear aggregation of the expanded polyalanine tract in the poly(A)-binding protein nuclear 1 (PABPN1) is the pathological hallmark of oculopharyngeal muscular dystrophy. However, wild type PABPN1 aggregates into nuclear inclusion in oxytocin-producing neurons under physiological conditions. In this study we have analyzed the nuclear organization and dynamics of PABPN1 inclusions in oxytocin-producing neurons. We demonstrated that PABPN1 inclusions represent a distinct compartment of the interchromatin region. They establish a spatial relationship with nuclear speckles, Cajal bodies and clastosomes. PABPN1 inclusions accumulate poly(A) RNA, but do not concentrate highly expressed mRNAs in oxytocin producing neurons and the mRNA-binding proteins hnRNP C, Y14 and REF. PABPN1 inclusions are dynamic structures that appear during the postnatal period and their number decrease in response to the activation of transcription. Our results support that the RNA retained in the PABPN1 inclusions is a noncoding regulatory RNA involved in some aspects of nuclear RNA metabolism. © 2007 Elsevier Inc. All rights reserved., This work was supported by the following grants: “Dirección General de Investigacion” of Spain (BFU2005-01030), “Fundación Marqués de Valdecilla” (API05/04) from Santander, Spain and “Centro de Investigación en Red sobre Enfermedades Neurodegenerativas” (CIBERNED, CB06/05/0037) from Spain.

Long-term survival remains low for most patients with glioblastoma (GBM), which reveals the need for markers of disease outcome and novel therapeutic targets. We describe that ODZ1 (also known as TENM1), a type II transmembrane protein involved in fetal brain development, plays a crucial role in the invasion of GBM cells. Differentiation of glioblastoma stem-like cells drives the nuclear translocation of an intracellular fragment of ODZ1 through proteolytic cleavage by signal peptide peptidase-like 2a. The intracellular fragment of ODZ1 promotes cytoskeletal remodelling of GBM cells and invasion of the surrounding environment both in vitro and in vivo. Absence of ODZ1 by gene deletion or downregulation of ODZ1 by small interfering RNAs drastically reduces the invasive capacity of GBM cells. This activity is mediated by an ODZ1-triggered transcriptional pathway, through the E-box binding Myc protein, that promotes the expression and activation of Ras homolog family member A (RhoA) and subsequent activation of Rho-associated, coiled-coil containing protein kinase (ROCK). Overexpression of ODZ1 in GBM cells reduced survival of xenografted mice. Consistently, analysis of 122 GBM tumour samples revealed that the number of ODZ1-positive cells inversely correlated with overall and progression-free survival. Our findings establish a novel marker of invading GBM cells and consequently a potential marker of disease progression and a therapeutic target in GBM., This work was supported by the Instituto de Salud Carlos III (ISCIII), grants PI13/01760 (JLF-L), PI12/00775 (PS-G) and SAF2014-53526-R (JL), and program Red Temática de Investigación Cooperativa en Cáncer (RTICC) grants RD12/0036/0022 (JLF-L), RD12/0036/0027 (PS-G), RD12/0036/0063 (JAM-C), RD12/0036/0003 (AP) and RD12-0036-0033 (JL), Instituto de Investigación Valdecilla (IDIVAL) grant APG/03 to JLF-L, and Cancer Research UK C33043/A12065 and Royal Society RG110591 to VS-M.

This paper studies the molecular organization, neuronal distribution and cellular differentiation dynamics of the giant fibrillar centers (GFCs) of nucleoli in rat sensory ganglia neurons. The GFC appeared as a round nucleolar domain (1–2 μm in diameter) partially surrounded by the dense fibrillar component and accompanied by numerous small FCs. By immunocytochemistry, the GFC concentrated the upstream binding factor, which may serve as a marker of this structure, and also contain RNA polymerase I, DNA topoisomerase I, SUMO-1 and Ubc9. However, they lack ubiquitin–proteasome conjugates and 20S proteasome. Transcription assay with 5′-fluorouridine incorporation revealed the presence of nascent RNA on the dense fibrillar component of the neuronal nucleolus, but not within the low electron-density area of the GFC. The formation of GFCs is neuronal size dependent: they were found in 58%, 30% and 0% of the large, medium and small neurons, respectively. GFCs first appeared during the postnatal period, concomitantly with a stage of neuronal growth, myelination and bioelectrical maturation. GFCs were not observed in segregated nucleoli induced by severe inhibition of RNA synthesis. We suggest that the formation of GFCs is associated with a high rate of ribosome biogenesis of the transcriptionally more active large-size neurons.

The neuron-like UR61 cell is a stable PC12 subline that contains a mouse N-ras oncogene. Dexamethasone (Dex) treatment induces a neuron-like differentiation, which is associated with neuritogenesis and nuclear expression of the glucocorticoid receptor and c-Jun. In differentiated UR61 cells, small ubiquitin-like modifiers 1 (SUMO-1) is concentrated in a new category of SUMO-1 nuclear bodies (SNBs) distinct from promyelocytic leukemia (PML) bodies by their large size and absence of PML protein. SNBs are 1 to 3 mum in diameter and exhibit a fine granular texture by electron microscopy. They are free of splicing factors and transcription foci and show spatial associations with Cajal bodies. In addition to SUMO-1 and the E2-conjugating enzyme Ubc9, which is essential for sumoylation, SNBs concentrate the transcriptional regulators CBP, CREB, and c-Jun. Moreover, transfection experiments demonstrate that SNBs accumulate the active conjugating form of SUMO-1 but not the conjugation defective variant of SUMO-1, supporting that SNBs are sites of sumoylation. Our results suggest that SNBs play a role in the control of the nucleoplasmic concentration of transcription regulators involved in neuroprotection and survival of the UR61 cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive weakness and muscle atrophy related to the loss of upper and lower motor neurons (MNs) without a curative treatment. There is experimental evidence suggesting that retinoids may be involved in ALS pathogenesis. Bexarotene (Bxt) is a retinoid-X receptor agonist used in the treatment of cutaneous lymphoma with a favorable safety profile whose effects have been recently investigated in other neurodegenerative diseases. In this study, we analyze the potential therapeutic effect of Bxt in the SOD1(G93A) mouse model of ALS. Mice were treated with Bxt or vehicle five times per week from day 60 onward. Survival, weight, and neuromuscular function studies together with histological and biochemical analyses were performed. Bxt significantly delayed motor function deterioration, ameliorated the loss of body weight, and extended mice survival up to 30% of the symptomatic period. Histological analyses of the lumbosacral spinal cord revealed that Bxt markedly delayed the early motor-neuron degeneration occurring at presymptomatic stages in ALS-transgenic mice. Bxt treatment contributed to preserve the MN homeostasis in the SOD1(G93A) mice. Particularly, it reduced the neuronal loss and the chromatolytic response, induced nucleolar hypertrophy, decreased the formation of ubiquitylated inclusions, and modulated the lysosomal response. As an agonist of the retinoic-X receptor (RXR) pathway, Bxt notably increased the nuclear expression of the RXRα throughout transcriptionally active euchromatin domains. Bxt also contributed to protect the MN environment by reducing reactive astrogliosis and preserving perisomatic synapsis. Overall, these neuroprotective effects suggest that treatment with Bxt could be useful in ALS, particularly in those cases related to SOD1 mutations. Acknowledgments. We thank Mrs Raquel Ceballos and Jana Arozamena for technical assistance. This work was supported by the following grants: “Dirección General de Investigación” of Spain (BFU2011-23983) and “Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED; CB06/05/0037)” from Spain

Chronic alcohol consumption may cause neurodevelopmental and neurodegenerative disorders. Alcohol neurotoxicity is associated with the production of acetaldehyde and reactive oxygen species that induce oxidative DNA damage. However, the molecular mechanisms by which ethanol disturbs the DNA damage response (DDR), resulting in a defective DNA repair, remain unknown. Here, we have used cultured primary cortical neurons exposed to 50 or 100 mM ethanol for 7 days to analyze the ethanol-induced DDR. Ethanol exposure produced a dose-dependent generation of double strand breaks and the formation of DNA damage foci immunoreactive for the histone gamma H2AX, a DNA damage marker, and for the ubiquitylated H2A, which is involved in chromatin remodeling at DNA damage sites. Importantly, these DNA damage foci failed to recruit the protein 53BP1, a crucial DNA repair factor. This effect was associated with a drop in 53BP1 mRNA and protein levels and with an inhibition of global transcription. Moreover, ethanol-exposed neurons treated with ionizing radiation (2 Gy) also failed to recruit 53BP1 at DNA damage foci and exhibited a greater vulnerability to DNA lesions than irradiated control neurons. Our results support that defective DNA repair, mediated by the deficient expression and recruitment of 53BP1 to DNA damage sites, represents a novel mechanism involved in ethanol neurotoxicity. The design of therapeutic strategies that increase or stabilize 53BP1 levels might potentially promote DNA repair and partially compensate alcohol neurotoxicity.

It is well known that the cell nucleus is organized in structural and functional compartments involved in transcription, RNA processing and protein modifications such as conjugation with SUMO-1 and proteolysis. Promyelocytic leukaemia (PML) bodies are dynamic nuclear structures that concentrate PML protein, SUMO-1 and several sumoylated and non-sumoylated protein regulators of nuclear functions. PML bodies and their associated CBP has been involved in neuronal survival. By light and electron microscopy immunocytochemistry and in situ hybridization we reported the presence, in non-pathological conditions, of a large PML-nuclear inclusion (PML-NI) in human supraoptic neurons. This inclusion appears as a single nuclear structure composed of a capsule enriched in PML, SUMO-1 and CBP proteins and a central lattice of filaments immunoreactive for class III β-tubulin, ubiquitinated proteins and proteasomes. Furthermore, the PML-NI concentrates the SUMO-conjugating enzyme E2 (UBC9). The PML-NI may be considered a nuclear factory involved in sumoylation and proteolysis via ubiquitin-proteasome system, two nuclear pathways engaged in the control of the nucleoplasmic concentration of active transcriptional regulators. Interestingly, the structural and molecular organization of the PML-NI is related to the Marinesco bodies, age-associated ubiquitinated intranuclear inclusions, and to the intranuclear rodlets enriched in class III β-tubulin, which are nuclear structures markedly decreased in Alzheimer's disease. © 2005 Elsevier Inc. All rights reserved., This study was supported by “Dirección General de Investigación Científica” from Spain (BFI2002-0454; BFU2005-01030), “Fondo de Investigación Sanitaria” from Spain (Red CIEN C03-C06) and Fundación Marqués de Valdecilla from Santander (API/05/04).

Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling pathways activated by EGFR and HER2 may help unravel novel therapeutic targets and molecular markers of response. Here, we show that treatment of breast cancer cell lines with blocking antibodies against EGFR (cetuximab) or HER2 (trastuzumab) promotes the specific induction of proapoptotic Bnip3L and chemosensitization. Moreover, we found that the Bnip3L gene is transcriptionally activated by FoxO3a. Trastuzumab-mediated induction of Bnip3L and nuclear translocation of FoxO3a was also shown in pleural effusion cells from a breast cancer patient. Transfection of breast cancer cells with constitutively active FoxO3a or with Bnip3L promotes sensitization to chemotherapy-induced apoptosis. On the contrary, blockade of Bnip3L expression by a small interfering RNA strategy significantly diminished the chemosensitizing effect of cetuximab. We found also an inverse correlation between EGFR and Bnip3L expression in surgical specimens from patients with breast cancer. Therefore, blockading EGFR or HER2 specifically up-regulates Bnip3L, which is required for chemosensitization of breast cancer cells. This novel pathway provides also the rationale for therapeutic strategies aimed to induce the expression of Bnip3L.

Acute inflammatory demyelinating polyneuropathy (AIDP) is a type of Guillain-Barré syndrome (GBS) characterized by primary nerve demyelination sometimes with secondary axonal degeneration. Studies on the fine structure of dorsal root ganglia in AIDP are lacking. Our aim was to investigate the cytology and nuclear organization of primary sensory neurons in AIDP with axonal injury using ultrastructural and immunohistochemical analysis. The light cytology of the L5 dorsal ganglion showed the characteristic findings of neuronal axonal reaction. The organization of chromatin, nucleolus, Cajal bodies, and nuclear pores corresponded to transcriptionally active neurons. However, the hallmark of the nuclear response to axonal injury was the formation of numerous nuclear bodies (NBs; 6.37 ± 0.6, in the AIDP, vs. 2.53 ± 0.2, in the control, mean ± SDM), identified as promyelocytic leukemia (PML) bodies by the presence of the protein PML. In addition to PML protein, nuclear bodies contained SUMO-1 and the transcriptional regulators CREB-binding protein (CBP) and glucocorticoid receptor (GR). The presence of proteasome 19S was also detected in some nuclear bodies. We suggest that neuronal PML bodies could regulate the nuclear concentration of active proteins, a process mediated by protein interactions with PML and SUMO-1 proteins. In the AIDP case, the proliferation of PML bodies may result from the overexpression of some nuclear proteins due to changes in gene expression associated with axonal injury. © 2004 Elsevier Inc. All rights reserved., This work was supported by the following grants: “Centro de Investigación de Enfermedades Neurológicas (CIEN)”, “Instituto de Salud Carlos III (Madrid, Spain)”; “Dirección General de Investigacion” from Spain (BFI2002-0454); and “Fundación Marqués de Valdecilla” (A04/03) from Santander, Spain.

Ras GTPases include the isoforms H-Ras, K-Ras, and N-Ras. Despite their great biochemical and biological similarities, evidence is mounting suggesting that Ras proteins may not be functionally redundant. A widespread strategy for studying small GTPases is the utilization of dominant inhibitory mutants that specifically block the activation of their respective wild-type proteins. As such, H-Ras N17 has proved to be extremely valuable as a tool to probe Ras functions. However, a comparative study on the inhibitory specificities of H-, K-, and N-Ras N17 mutants has not been approached thus far. Herein, we demonstrate that H-, K-, and N-Ras N17 mutants exhibit markedly distinct inhibitory effects toward H-, K-, and N-Ras. H-Ras N17 can effectively inhibit the activation of all three isoforms. K-Ras N17 completely blocks the activation of K-Ras and is only slightly inhibitory on H-Ras. N-Ras N17 can mainly inhibit N-Ras activation. In light of the recent data on the compartmentalization of H-Ras and K-Ras in the plasma membrane, here we present for the first time a description of N-Ras cellular microlocalization. Overall, our results on Ras N17 mutants specificities exhibit a marked correlation with the localization of the Ras isoforms to distinct membrane microdomains.

The cell nucleus is divided into chromosome territories and the extrachromosomal domain. The latter includes several structural and functional compartments involved in RNA processing and transport. Morphological and cytochemical analyses of the cell nucleus in sturgeon podocytes revealed the existence of a previously uncharacterised nuclear compartment. It appears as a cleared nucleoplasmic area of variable size within the extrachromosomal domain. Conventional light and electron microscopy revealed that this compartment, here referred to as cleared extrachromosomal domain (CED), appears free of chromatin and RNA-containing organelles and is closely surrounded by heterochromatin masses. Cytochemical and immunogold electron microscope studies indicated that CEDs lack DNA, RNA and glycoconjugates. The ultrastructural examination of Lowicryl-embedded sections showed that CEDs are formed by a fibrillar network. In resinless preparations, this network appears as a dense mesh of crosslinked nuclear matrix filaments. The density of nuclear matrix filaments within the CED is remarkably higher than that observed in the rest of the nucleus. Our results indicate that the CED is a single, distinct extrachromosomal domain of the nuclear matrix of sturgeon podocytes. The absence in the CED of detectable DNA and RNA, as well as the lack of chromatin and RNA-containing structures, suggests that transcription and RNA processing do not occur in this novel nuclear compartment. The volume occupied by the CED may preserve the volume-to-cytoplasm ratio in the podocyte and contribute to modulate the spatial organisation and the volume occupied by the chromosomal territories.

It is well established that forskolin-induced elevation of cAMP results in activation of DNA synthesis in Schwann cell cultures. This promitotic response is partially mediated by the Cdk2, which is required for the transition from the G1 to the S phase of the cell cycle. In the present study, we analyze the effects of cAMP elevation in cultured Schwann cells on the transcriptional activity and on the organization of two nuclear compartments involved in pre-mRNA processing: Cajal bodies (CBs) and splicing factor compartments. Our immunofluorescence and quantitative studies show that forskolin treatment induces a 5.6-fold increase in the proportion of S phase Schwann cells, detected by a short pulse (20 min) of BrdU incorporation. This increase in DNA synthesis correlates with an activation of global transcription, as is indicated by the higher nuclear incorporation of BrU in nascent RNA. Forskolin treatment significantly increases the percentage of Schwann cells containing typical CBs, which concentrate spliceosomal snRNPs and the survival motor neuron (SMN) protein. This increase in the number of CBs closely correlates with the activation of transcription. Moreover, the occurrence of CBs is significantly higher in BrdU (+) cells than in BrdU (-) cells, indicating that entry in the S phase promotes the formation of CBs. During the S phase, Schwann cell nuclei display higher Cdk2 nuclear staining and concentrate this kinase in CBs. Forskolin also induces a redistribution of the pre-mRNA splicing factors in Schwann cells. Primary cultures of Schwann cells provide an excellent physiological model to demonstrate that the assembly of CBs is a transcription- and replication-dependent cellular event. Moreover, the S phase accumulation of Cdk2 observed in Schwann cells supports a functional link between CBs and DNA replication, which is mediated by the possible participation of CBs in the regulatory control of histone gene expression.

The proteasome is responsible for most of the protein degradation that takes place in the cytoplasm and nucleus. Immunofluorescence and electron microscopy are used to study proteasome dynamics during the cell cycle in rat Schwann cells. During interphase, the proteasome is present in the nucleus and cytoplasm and shows no colocalization with cytoskeletal components. Some cytoplasmic proteasomes always localize in the centrosome both in interphase and in mitotic cells and only associate with microtubules during mitosis. The proteasome exits the nucleus during prophase. In anaphase, the proteasome becomes prominent in the region between the two sets of migrating chromosomes and in association with interzonal microtubules and stem bodies. In telophase, the proteasome begins to reenter the nucleus and is prominent in the midbody region until the end of cytokinesis. The proteasome does not colocalize with actin or vimentin during mitosis, except for colocalization with actin in the sheet-like lamellipodia, which serve as substrate attachments for the cell during mitosis. During S phase, nuclear proteasomes colocalize with foci of BrdU incorporation, but this association changes with time: maximal at early S phase and declining as S phase progresses to the end. These results are discussed in relation to the biochemical pathways involved in cell cycle progression.

It is well-established that the neuronal cell nucleus is organized in discrete compartments involved in transcription and RNA processing. The main nuclear compartments in neurons include the chromosome territories, the nucleolus, nuclear speckles of splicing factors, Cajal bodies, and nuclear rodlets. The supraoptic nucleus (SON) neurons provide a powerful model in vivo to study the organization of these nuclear compartments in response to variations of cellular activity. The upregulation of transcription in SON neurons under chronic hyperosmolar conditions is associated with 1) nuclear and nucleolar enlargement, 2) dispersion of chromatin, 3) reduction in the size of nuclear speckles, 4) increase in the number of Cajal bodies implicated in the maturation of splicing small nuclear ribonucleoproteins, and 5) proliferation of the fibrillar centers of the nucleolus, the sites of nucleolar transcription of ribosomal genes. These changes revert after the cessation of the activation by rehydration of animals. Under conditions of neuronal stress induced by hypertonic saline injection, SON neurons exhibit an early response of downregulation of transcription. This is accompanied by chromatin condensation, redistribution of splicing factors, reduction in the number of Cajal bodies, and microsegregation of the fibrillar and granular components of the nucleolus and disruption of its fibrillar centers, all of which are associated with a transitory expression of c-Fos. These changes progressively revert and at 24 hours after the stress induction a rebound upregulation of transcription is observed. These findings illustrate the transcription-dependent organization and behavior of nuclear compartments in the neuronal model of magnocellular neurosecretory cells of the hypothalamus.

Nuclear architecture is highly concerted including the organization of chromosome territories and distinct nuclear bodies, such as nucleoli, Cajal bodies, nuclear speckles of splicing factors, and promyelocytic leukemia nuclear bodies, among others. The organization of such nuclear compartments is very dynamic and may represent a sensitive indicator of the functional status of the cell. Here, we describe methodologies that allow isolating discrete cell populations from the brain and the fine observation of nuclear signs that could be insightful predictors of an early neuronal injury in a wide range of neurodegenerative disorders. The tools here described may be of use for the early detection of pre-degenerative processes in neurodegenerative diseases and for validating novel rescue strategies.

We investigated neuronal self-defense mechanisms in a murine model of amyotrophic lateral sclerosis (ALS), the transgenic hSOD1(G93A), during both the asymptomatic and symptomatic stages. This is an experimental model of endoplasmic reticulum (ER) stress with severe chromatolysis. As a compensatory response to translation inhibition, chromatolytic neurons tended to reorganize the protein synthesis machinery at the perinuclear region, preferentially at nuclear infolding domains enriched in nuclear pores. This organization could facilitate nucleo-cytoplasmic traffic of RNAs and proteins at translation sites. By electron microscopy analysis, we observed that the active euchromatin pattern and the reticulated nucleolar configuration of control motor neurons were preserved in ALS chromatolytic neurons. Moreover the 5'-fluorouridine (5'-FU) transcription assay, at the ultrastructural level, revealed high incorporation of the RNA precursor 5'-FU into nascent RNA. Immunogold particles of 5'-FU incorporation were distributed throughout the euchromatin and on the dense fibrillar component of the nucleolus in both control and ALS motor neurons. The high rate of rRNA transcription in ALS motor neurons could maintain ribosome biogenesis under conditions of severe dysfunction of proteostasis. Collectively, the perinuclear reorganization of protein synthesis machinery, the predominant euchromatin architecture, and the active nucleolar transcription could represent compensatory mechanisms in ALS motor neurons in response to the disturbance of ER proteostasis. In this scenario, epigenetic activation of chromatin and nucleolar transcription could have important therapeutic implications for neuroprotection in ALS and other neurodegenerative diseases. Although histone deacetylase inhibitors are currently used as therapeutic agents, we raise the untapped potential of the nucleolar transcription of ribosomal genes as an exciting new target for the therapy of some neurodegenerative diseases. Acknowledgements: The authors wish to thank Raquel García-Ceballos for technical assistance. This work was supported by the following grants: “Dirección General de Investigación” of Spain (BFU2011-23983), and “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; CB06/05/0037)” from Spain

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called “residual” CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

Administration of tellurium (Te) in weaning rats causes a well-established demyelinating neuropathy induced by the inhibition in myelinating Schwann cells (SC) of the synthesis of cholesterol, a major component of the myelin sheath, at the level of squalene epoxidase. We have used this experimental model of Te neuropathy to study the biogenesis and reorganization of the endomembranes of the nuclear envelope and endoplasmic reticulum (ER) in response to Te treatment by ultrastructural analysis and in situ hybridization for the detection of HMG CoA reductase and synthase mRNA, which encode key enzymes in cholesterol synthesis. The adaptive response of myelinating SC to cholesterol depletion includes cell hypertrophy, the formation of tubular invaginations of proliferating nuclear membranes giving rise to peculiar nuclear inclusions termed crystalloids, and, at the cytoplasmic level, the formation of lamellar bodies of rough ER, proliferation of the smooth ER, and overexpression of HMG CoA reductase and synthase mRNAs. The changes revert after withdrawal of Te treatment. Our results show that the biogenesis and structural organization of both endomembrane systems change dynamically upon Te-induced cholesterol depletion, indicating that this constituent plays a critical role in the organization of nuclear envelope and ER compartments in SC. The results also suggest that the HMG CoA reductase, an integral membrane protein of ER, provides the signal for the extensive membrane assembly. While the physiological meaning of crystalloid remains to be clarified, the hypertrophy of the smooth ER may represent a cytoprotective mechanism involved in detoxification of the neurotoxic agent or its metabolic derivates. GLIA 29:246–259, 2000. © 2000 Wiley-Liss, Inc.

We have used an experimental model of tellurium (Te)-induced demyelinating neuropathy in the rat to study cellular mechanisms involved in the early response of myelinating Schwann cells (SCs) to injury, prior to demyelination. Starting at postnatal day 21, weaned rats were fed a diet containing 1.1% elemental Te. The animals were killed daily within the 1st week of Te diet and the sciatic nerves were processed for the ultrastructural and immunocytochemical studies. Immunohistochemistry revealed that Te induces an increased nuclear expression of c-Fos in SCs. By electron microscopy analysis, the early cytoplasmic alteration was a dramatic disorganization of the rough endoplasmic reticulum (ER) with cisternal dilations and redistribution and loss of membrane-bound ribosomes. This was followed by a prominent activation of the macroautophagy in SCs. This process involved the formation of autophagosomes containing well-preserved cell organelles, autolysosomes with cellular remnants in various phases of degeneration and lysosomes. Te treatment also induced the expression of free ubiquitin in the perikaryal region of the SC cytoplasm. Immunogold electron microscopy showed the subcellular distribution of ubiquitin in the cytosol, around of dilated ER cisterns and in the matrix of autolysosomes and residual bodies. At the nucleolar level, fibrillarin immunofluorescence revealed nucleolar segregation in SCs exposed to Te. The ultrastructural study confirmed the segregation of the nucleolar components with a peripheral distribution of the dense fibrillar component. These results support the hypothesis that the depletion of cholesterol induced by Te treatment triggers a stress response in myelinating SCs mediated by immediate early genes of the fos family. The cellular response includes a severe disruption of the protein synthesis machinery, namely the rough ER and nucleolus, with the subsequent activation of both ubiquitin and autophagic pathways of proteins and cell organelle degradation. This cytoplasmic remodeling may represent a cytoprotective mechanism in the response of SCs to a neurotoxic stress. Furthermore, it must be a prerequisite for the induction of phenotypic changes and cell repair mechanisms in SCs.