Filipe Monteiro

Activation of ERK1/2 MAP kinases in Familial Amyloidotic Polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by the extracellular deposition of transthyretin (TTR), especially in the PNS. Given the invasiveness of nerve biopsy, salivary glands (SG) from FAP patients were used previously in microarray analysis; mitogen‐activated protein (MAP) kinase phosphatase 1 (MKP‐1) was down‐regulated in FAP. Results were validated by RT‐PCR and immunohistochemistry both in SG and in nerve biopsies of different stages of disease progression. MKP‐3 was also down‐regulated in FAP SG biopsies. Given the relationship between MKPs and MAPKs, the latter were investigated. Only extracellular signal‐regulated kinases 1/2 (ERK1/2) displayed increased activation in FAP SG and nerves. ERK1/2 kinase (MEK1/2) activation was also up‐regulated in FAP nerves. In addition, an FAP transgenic mouse model revealed increased ERK1/2 activation in peripheral nerve affected with TTR deposition when compared to control animals. Cultured rat Schwannoma cell line treatment with TTR aggregates stimulated ERK1/2 activation, which was partially mediated by the receptor for advanced glycation end‐products (RAGE). Moreover, caspase‐3 activation triggered by TTR aggregates was abrogated by U0126, a MEK1/2 inhibitor, indicating that ERK1/2 activation is essential for TTR aggregates‐induced cytotoxicity. Taken together, these data suggest that abnormally sustained activation of ERK in FAP may represent an early signaling cascade leading to neurodegeneration.

Several Cis-regulatory Elements Control mRNA Stability, Translation Efficiency, and Expression Pattern of Prrxl1 (Paired Related Homeobox Protein-like 1)

Background: The mechanisms that control the Prrxl1 expression are poorly understood. Results: Several regulatory elements present in Prrxl1 alternative promoters are functionally characterized, including a binding motif for Phox2b required for Prrxl1 expression in visceral sensory neurons. Conclusion: We define diverse regulatory modules, which control the spatiotemporal expression of Prrxl1 in nociceptive neurons. Significance: A new mechanism involved in the ganglion specific action of Prrxl1 is described. The homeodomain transcription factor Prrxl1/DRG11 has emerged as a crucial molecule in the establishment of the pain circuitry, in particular spinal cord targeting of dorsal root ganglia (DRG) axons and differentiation of nociceptive glutamatergic spinal cord neurons. Despite Prrxl1 importance in the establishment of the DRG-spinal nociceptive circuit, the molecular mechanisms that regulate its expression along development remain largely unknown. Here, we show that Prrxl1 transcription is regulated by three alternative promoters (named P1, P2, and P3), which control the expression of three distinct Prrxl1 5′-UTR variants, named 5′-UTR-A, 5′-UTR-B, and 5′-UTR-C. These 5′-UTR sequences confer distinct mRNA stability and translation efficiency to the Prrxl1 transcript. The most conserved promoter (P3) contains a TATA-box and displays in vivo enhancer activity in a pattern that overlaps with the zebrafish Prrxl1 homologue, drgx. Regulatory modules present in this sequence were identified and characterized, including a binding site for Phox2b. Concomitantly, we demonstrate that zebrafish Phox2b is required for the expression of drgx in the facial, glossopharyngeal, and vagal cranial ganglia.

Ser119 phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor

PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1(-/-) mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, whereas the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified, Ser¹¹⁹ and Ser²³⁸. Phosphorylation at Ser¹¹⁹ is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser¹¹⁹ phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.

Paired related homeobox protein-like 1 (Prrxl1) controls its own expression by a transcriptional autorepression mechanism.

The homeodomain factor paired related homeobox protein‐like 1 (Prrxl1) is crucial for proper assembly of dorsal root ganglia (DRG)–dorsal spinal cord (SC) pain‐sensing circuit. By performing chromatin immunoprecipitation with either embryonic DRG or dorsal SC, we identified two evolutionarily conserved regions (i.e. proximal promoter and intron 4) of Prrxl1 locus that show tissue‐specific binding of Prrxl1. Transcriptional assays confirm the identified regions can mediate repression by Prrxl1, while gain‐of‐function studies in Prrxl1 expressing ND7/23 cells indicate Prrxl1 can down‐regulate its own expression. Altogether, our results suggest that Prrxl1 uses distinct regulatory regions to repress its own expression in DRG and dorsal SC.

Isolated ventricular noncompaction: a case report

Isolated ventricular noncompaction is an extremely rare cardiomyopathy, not fully clarified.It is characterized by persistent embryonic myocardium morphology without associated cardiac abnormalities.Since first description in 1984, few clinical studies were done. Data in the literature are lacking and most reports consist on a few case studies.Doppler ecocardiogram is considered the reference method for diagnosis.Diagnosis remains difficult since there are similarities with other cardiac defects, clinical manifestations are non-specific and echocardiographic criteria are not universally accepted.As a consequence diagnosis may be easily missed.Moreover, clinical and echocardiographic features were just recently clarified.Treatment is directed towards important clinical manifestations (heart failure, arrhythmias and embolic events).We present a clinical case of severe cardio-respiratory failure in previously healthy and asymptomatic young male, which was the initial presentation of an isolated ventricular noncompaction.A brief review of available literature is done concerning to this case study.

Dual role of Tlx3 as modulator of Prrxl1 transcription and phosphorylation

The proper establishment of the dorsal root ganglion/spinal cord nociceptive circuitry depends on a group of homeodomain transcription factors that includes Prrxl1, Brn3a and Tlx3. By the use of epistatic analysis, it was suggested that Tlx3 and Brn3a, which highly co-localize with Prrxl1 in these tissues, are required to maintain Prrxl1 expression. Here, we report two Tlx3-dependent transcriptional mechanisms acting on Prrxl1 alternative promoters, referred to as P3 and P1/P2 promoters. We demonstrate that (i) Tlx3 induces the transcriptional activity of the TATA-containing promoter P3 by directly binding to a bipartite DNA motif and (ii) it synergistically interacts with Prrxl1 by indirectly activating the Prrxl1 TATA-less promoters P1/P2 via the action of Brn3a. The Tlx3 N-terminal domain 1-38 was shown to have a major role on the overall Tlx3 transcriptional activity and the C-terminus domain (amino acids 256-291) to mediate the Tlx3 effect on promoters P1/P2. On the other hand, the 76-111 domain was shown to decrease Tlx3 activity on the TATA-promoter P3. In addition to its action on Prrxl1 alternative promoters, Tlx3 proved to have the ability to induce Prrxl1 phosphorylation. The Tlx3 domain responsible for Prrxl1 hyperphosphorylation was mapped and encompasses amino acid residues 76 to 111. Altogether, our results suggest that Tlx3 uses distinct mechanisms to tightly modulate Prrxl1 activity, either by controlling its transcriptional levels or by increasing Prrxl1 phosphorylation state.

Relatório de actividades da UCIR – 1994

RESUMO Desde 1990, com a informatizacao dos principais dados clinicos dos doentes para os quais foi feito um software adequado, a Unidade de Cuidados Intensivos Respiratorios, passou a elaborarum relatorio anual da actividade assistencial, pedagogica e de investigacao. Nestes relatorios, para alem da analise estatistica dos varios dados, sao avaliados os resultados em relacao aos objectivos programados. No presente relatorio foi ainda analisada a evolucao do internamento desde a abertura da Unidade, bem como o Follow Up dos doentes apos a alta e proposto um protocolo para ventilacao domiciliaria que resultou da experiencia adquirida pela UCIR ao longo dos ultimos anos.

Zinc finger transcription factor Casz1 expression is regulated by homeodomain transcription factor Prrxl1 in embryonic spinal dorsal horn late-born excitatory interneurons.

The transcription factor Casz1 is required for proper assembly of vertebrate vasculature and heart morphogenesis as well as for temporal control of Drosophila neuroblasts and mouse retina progenitors in the generation of different cell types. Although Casz1 function in the mammalian nervous system remains largely unexplored, Casz1 is expressed in several regions of this system. Here we provide a detailed spatiotemporal characterization of Casz1 expression along mouse dorsal root ganglion (DRG) and dorsal spinal cord development by immunochemistry. In the DRG, Casz1 is broadly expressed in sensory neurons since they are born until perinatal age. In the dorsal spinal cord, Casz1 displays a more dynamic pattern being first expressed in dorsal interneuron 1 (dI1) progenitors and their derived neurons and then in a large subset of embryonic dorsal late‐born excitatory (dILB) neurons that narrows gradually to become restricted perinatally to the inner portion. Strikingly, expression analyses using Prrxl1‐knockout mice revealed that Prrxl1, a key transcription factor in the differentiation of dILB neurons, is a positive regulator of Casz1 expression in the embryonic dorsal spinal cord but not in the DRG. By performing chromatin immunoprecipitation in the dorsal spinal cord, we identified two Prrxl1‐bound regions within Casz1 introns, suggesting that Prrxl1 directly regulates Casz1 transcription. Our work reveals that Casz1 lies downstream of Prrxl1 in the differentiation pathway of a large subset of dILB neurons and provides a framework for further studies of Casz1 in assembly of the DRG–spinal circuit.

Ventilação mecânica e obstinação terapêutica ou distanásia, a dialéctica da alta tecnologia em medicina intensiva

Resumo Distanasia ou qualquer um dos seus sinonimos e uma consequencia do excesso terapeutico em relacao ao prognostico esperado. A obstinacao terapeutica e um dos dilemas eticos mais angustiantes no quotidiano de medicina intensiva, apesar de a sua apreciacao encontrar um suporte normativo em varias instituicoes e organizacoes. A manutencao ou nao suspensao da ventilacao mecânica numa determinada circunstância de fim de vida pode ser considerado como um exemplo paradigmatico de obstinacao terapeutica. A compreensao desta postura passa pela analise e reflexao do acto medico a luz de alguns conceitos etico-filosoficos.

A role for Prolyl-isomerase PIN1 in the phosphorylation-dependent modulation of PRRXL1 function

Prrxl1 encodes for a paired-like homeodomain transcription factor essential for the correct establishment of the dorsal root ganglion - spinal cord nociceptive circuitry during development. Prrxl1-null mice display gross anatomical disruption of this circuitry, which translates to a markedly diminished sensitivity to noxious stimuli. Here, by the use of an immunoprecipitation and mass spectrometry approach, we identify five highly conserved phosphorylation sites (T110, S119, S231, S233 and S251) in PRRXL1 primary structure. Four are phospho-S/T-P sites, which suggest a role for the prolyl isomerase PIN1 in regulating PRRXL1. Accordingly, PRRXL1 physically interacts with PIN1 and displays diminished transcriptional activity in a Pin1-null cell line. Additionally, these S/T-P sites seem to be important for PRRXL1 conformation, and their point mutation to alanine or aspartate down-regulates PRRXL1 transcriptional activity. Altogether, our findings provide evidence for a putative novel role of PIN1 in the development of the nociceptive system and indicate phosphorylation-mediated conformational changes as a mechanism for regulating the PRRXL1 role in the process.