RBM45 homo-oligomerization mediates association with ALS-linked proteins and stress granules.

The aggregation of RNA-binding proteins is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RBM45 is an RNA-binding protein that forms cytoplasmic inclusions in neurons and glia in ALS and FTLD. To explore the role of RBM45 in ALS and FTLD, we examined the contribution of the protein's domains to its function, subcellular localization, and interaction with itself and ALS-linked proteins. We find that RBM45 forms homo-oligomers and physically associates with the ALS-linked proteins TDP-43 and FUS in the nucleus. Nuclear localization of RBM45 is mediated by a bipartite nuclear-localization sequence (NLS) located at the C-terminus. RBM45 mutants that lack a functional NLS accumulate in the cytoplasm and form TDP-43 positive stress granules. Moreover, we identify a novel structural element, termed the homo-oligomer assembly (HOA) domain, that is highly conserved across species and promote homo-oligomerization of RBM45. RBM45 mutants that fail to form homo-oligomers exhibit significantly reduced association with ALS-linked proteins and inclusion into stress granules. These results show that RMB45 may function as a homo-oligomer and that its oligomerization contributes to ALS/FTLD RNA-binding protein aggregation.

Alterations of Ca²âº-responsive proteins within cholinergic neurons in aging and Alzheimer's disease.

The molecular basis of selective neuronal vulnerability in Alzheimer's disease (AD) remains poorly understood. Using basal forebrain cholinergic neurons (BFCNs) as a model and immunohistochemistry, we have demonstrated significant age-related loss of the calcium-binding protein calbindin-D(28K) (CB) from BFCN, which was associated with tangle formation and degeneration in AD. Here, we determined alterations in RNA and protein for CB and the Ca(2+)-responsive proteins Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), growth-associated protein-43 (GAP43), and calpain in the BF. We observed progressive downregulation of CB and CaMKI RNA in laser-captured BFCN in the normal-aged-AD continuum. We also detected progressive loss of CB, CaMKIδ, and GAP43 proteins in BF homogenates in aging and AD. Activated µ-calpain, a calcium-sensitive protease that degrades CaMKI and GAP43, was significantly increased in the normal aged BF and was 10 times higher in AD BF. Overactivation of µ-calpain was confirmed using proteolytic fragments of its substrate spectrin. Substantial age- and AD-related alterations in Ca(2+)-sensing proteins most likely contribute to selective vulnerability of BFCN to degeneration in AD.

Neurodegenerative diversity in human cortical contusion: histological analysis of tissue derived from decompressive craniectomy.

The principal aim in the management of patients with cerebral contusion (CC) following severe traumatic brain injury (TBI) is the prevention, amelioration, and treatment of secondary neuronal dysfunction and pathology. Distinguishing between irreversibly damaged and surviving tissue could have considerable therapeutic and prognostic implications for patients. To characterize structurally the neuronal compartment of the contused region in samples derived from patients who suffered severe TBI and were subjected to decompressive craniectomy, we used NeuN, a neuronal marker. We determined that NeuN "patches", sectors with loss of NeuN immunoreactivity (NeuN-IR), represented 25% of the area among the analyzed cases. We also found a 67% decrease in NeuN levels via Western blot. Tissue adjoining patches of NeuN-IR were considered "preserved" due to the apparent normal density of neurons and conservation of the six cortical layers. Nevertheless, these sectors retained only 39% of their neurons with the classical pattern described for normal NeuN-IR. Using Fluorojade we identified a 16-fold increase in density of moribund neurons in "preserved" sectors when compared to controls. Additionally these abnormalities were enhanced 5-fold in "patches" of NeuN-IR when compared to preserved regions. Therefore, NeuN/Fluorojade abnormalities are indicative of different cell fates characteristic of CC tissue. This analysis addressed exclusively the neuronal compartment and provides new insights into the degenerative state of neurons in the contused region that is likely to contribute to clinical outcome and differentiate TBI from ischemia.

The RNA-binding motif 45 (RBM45) protein accumulates in inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) patients.

RNA-binding protein pathology now represents one of the best characterized pathologic features of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration patients with TDP-43 or FUS pathology (FTLD-TDP and FTLD-FUS). Using liquid chromatography tandem mass spectrometry, we identified altered levels of the RNA-binding motif 45 (RBM45) protein in the cerebrospinal fluid (CSF) of ALS patients. This protein contains sequence similarities to TAR DNA-binding protein 43 (TDP-43) and fused-in-sarcoma (FUS) that are contained in cytoplasmic inclusions of ALS and FTLD-TDP or FTLD-FUS patients. To further characterize RBM45, we first verified the presence of RBM45 in CSF and spinal cord tissue extracts of ALS patients by immunoblot. We next used immunohistochemistry to examine the subcellular distribution of RBM45 and observed in a punctate staining pattern within nuclei of neurons and glia in the brain and spinal cord. We also detected RBM45 cytoplasmic inclusions in 91 % of ALS, 100 % of FTLD-TDP and 75 % of Alzheimer's disease (AD) cases. The most extensive RBM45 pathology was observed in patients that harbor the C9ORF72 hexanucleotide repeat expansion. These RBM45 inclusions were observed in spinal cord motor neurons, glia and neurons of the dentate gyrus. By confocal microscopy, RBM45 co-localizes with ubiquitin and TDP-43 in inclusion bodies. In neurons containing RBM45 cytoplasmic inclusions we often detected the protein in a punctate pattern within the nucleus that lacked either TDP-43 or ubiquitin. We identified RBM45 using a proteomic screen of CSF from ALS and control subjects for candidate biomarkers, and link this RNA-binding protein to inclusion pathology in ALS, FTLD-TDP and AD.

Age-related loss of calcium buffering and selective neuronal vulnerability in Alzheimer's disease.

The reasons for the selective vulnerability of distinct neuronal populations in neurodegenerative disorders are unknown. The cholinergic neurons of the basal forebrain are vulnerable to pathology and loss early in Alzheimer's disease and in a number of other neurodegenerative disorders of the elderly. In the primate, including man, these neurons are rich in the calcium buffer calbindin-D(28K). Here, we confirm that these neurons undergo a substantial loss of calbindin in the course of normal aging and report a further loss of calbindin in Alzheimer's disease both at the level of RNA and protein. Significantly, cholinergic neurons that had lost their calbindin in the course of normal aging were those that selectively degenerated in Alzheimer's disease. Furthermore, calbindin-containing neurons were virtually resistant to the process of tangle formation, a hallmark of the disease. We conclude that the loss of calcium buffering capacity in these neurons and the resultant pathological increase in intracellular calcium are permissive to tangle formation and degeneration.

Caracterización de un modelo organotípico de cultivos de neuronas corticales de humano derivadas de trauma craneoencefalico / Organotypic human neuronal culture derived from traumatic brain injury

Introducción: El trauma craneoencefalico (TEC) es un problema de salud global que puede generar en los pacientes que lo padecen, muerte, discapacidad y/o alteraciones psiquißtricas con gran impacto sobre su desempe±o posterior y sobre su ßmbito familiar. En los últimos años se ha avanzado en el conocimiento de los mecanismos fisiopatológicos que subyacen al TCE. Sin embargo, esto no estß completamente entendido, como tampoco hay claridad sobre los mecanismos de neuroprotección. Por esta razón cada vez mas se buscan modelos que permitan aproximarse al estudio de este síndrome y de esta manera aproximarse a la neuroprotección. Objetivo: Caracterizar un modelo de cultivo organotípico de neuronas corticales humanas obtenidas de personas que sufrieron TCE y a las cuales se les practicó remoción de la contusión. Metodología: Se utilizó tejido cortical humano procedente de 4 individuos que sufrieron TCE y a los cuales se les removió la contusión. Se obtuvieron tajadas de corteza cerebral de 1,500-2,000 mm, las cuales se mantuvieron en un flujo continuo de LCRa a 2 ml/min y una mezcla gaseosa de O2 al 95 por ciento y CO2 al 5 por ciento con burbujeo permanente durante 2, 8 y 14 horas. Se tomó como tiempo cero el momento de obtención de la muestra. Después de cada tiempo se tomaron las tajadas, se cortaron en un vibratomo de medio líquido a 50 mm y se procesaron inmunohistoquímicamente con los marcadores neuronales de degeneración NeuN y MAP2. Resultados: Los resultados indican que las muestras de corteza cerebral se pudieron mantener con cierto grado de integridad celular y laminar hasta las 2 horas de cultivo. Se observó que a partir de este tiempo se inicia un proceso de alteración de la citoarquitectura neuronal y laminar, determinada por la pérdida y alteración de la inmunorreactividad a los marcadores NeuN y MAP2. Ademas se encontró que hay vulnerabilidad celular que compromete en mayor medida a las neuronas localizadas en las laminas corticales III y V.

Introduction: Traumatic brain injury is a global medical problem whose survivors may show disability and neurological or psychiatric sequelae. In the last few years the knowledge of physiopathological mechanisms of TBI has increase but still it is not entirely known. For this reason the research has turn over in one´s mind in new strategies to study this pathology looking for neuroprotection. Objective: The aim of this work is to develop an organotypic culture of cortical human neurons derived from a contusion tissue obtain from patients that suffered TBI. Methodology: We used contused brain tissue from 4 TBI patients. Sections between 1,500-2,000 mm were kept in a continuous flow of aCSF 2 ml/min in a mixture of 95 percent O2 and 5 percent CO2 for 2, 8 and 14 hours. The initial time (0 hours) was the tissue extraction time. From blocks, sections of 50 mm were obtained and processed for immunocytochemistry to NeuN and MAP2. Results: The results show that organotypic cultures keep neuron integrity and laminar organization in the cerebral cortex slices from 0 to 2 hours. From this time ahead neuronal morphology and laminar organization is altered especially in neurons located on layers III and V.

Alteración de la organización laminar y de la dendroarquitectura de la corteza cerebral del humano post-trauma craneoencefalico / Changes in laminar and dendritic organization of the cerebral cortex after traumatic brain injury

Introducción: El trauma craneoencefalico (TCE)es un fenómeno heterogéneo desde el punto de vista molecular, celular y en la respuesta clínica. Se considera que esta diversidad se debe a la intensidad de la injuria primaria, eventos secundarios asociados (hipoxia, isquemia, edema, inflamación), al estado metabólico del paciente, su base genética, edad, género, etc. Para determinar la integridad anatomo-funcional de las células nerviosas es importante verificar el estado de la cito, dendroarquitectura y preservación laminar como un requisito para garantizar conectividad. Objetivo: Valorar la respuesta de las neuronas al trauma con dos marcadores neuronales selectivos sensibles a la lesión NeuN y MAP2. Materiales y métodos: Se utilizaron muestras (4 de lóbulo temporal y 2 de lóbulo frontal) de 6 pacientes que habían sufrido TCE. Las muestras se fijaron en PLP, cortadas en vibrßtomo a 50 µm, incubadas con los anticuerpos NeuN y MAP2 y procesadas con el sistema avidina-biotina. Como control se utilizó tejido humano post-mortem. Resultados: La inmunorreactividad (IR) para NeuN fue anormal en todas las muestras, con sectores que mostraron IR ligeramente alterada, otros con perdida parcial de las capas supragranulares, sobre todo la lßmina III y otros con pérdida drastica de todas las laminas. La IR para MAP2 se alteró en todas las muestras con diferentes grados de compromiso. Los procesos dendríticos fueron difíciles de seguir, especialmente los procedentes de la lßmina V, los cuales se observaron tortuosos, fragmentados y con orientación aberrante. Conclusiones: Con el propósito de conocer el estado de las neuronas después de un evento lesivo se recomienda el uso de los marcadores NeuN y MAP2 complementarios a los métodos clasicos. El presente trabajo muestra la diversidad de respuestas histopatológicas en sectores adyacentes de una misma muestra con ambos marcadores, como un indicador de los diferentes estados de neurodegeneración.

Introduction: Traumatic brain injury (TBI) is a heterogeneous phenomenon from a molecular, cellular and pathological perspective. Clinical outcome is also extremely variable. It is considered that such a diversity response to TBI is related to the primary injury intensity, associated secondary events (hypoxia, ischemia, oedema and inflammation), metabolic patient state, genetic background, age, gender, etc. After injury the histopathological outcome is variable in time and space. In order to determine the anatomofunctional integrity of nerve cells in the cerebral cortex, it is important to verify the state of the cito and dendroarchitecture and the laminar preservation as a requisite to guarantee connectivity. Objective: The aim of the present work was to evaluate the response of human cortical neurons using two selective neuronal markers, NeuN and MAP2, which recognize citoarchitecture and dendritic arrangement, respectively. Materials and methods: In the present study we utilized six tissue samples (4 temporal and 2 frontal cortices) from TBI patients. Tissues from four post-mortem human brains were used as controls. Tissue samples were fixed in PLP, cut at 50 um in a vibratome, incubated with NeuN and MAP2 and processes with the avidin/biotin complex. Results: NeuN-IR was abnormal in all samples analyzed with some sectors showing slight NeuN-IR, others with NeuN-IR partial loss in supragranular layers, especially layer IIII, and other with a drastic reduction in staining in all cortical layers. MAP2-IR was altered across sections with sectors showing different degrees of changes in the normal pattern of MAP2-IR. Dendritic processes were difficult to follow because of its discontinuity. Layer V apical dendritic processes appear tortuous and its IR was fragmented in some cases they take aberrant orientations.