Rodrigo A. P. Martins

Implications of aneuploidy for stem cell biology and brain therapeutics

Understanding the cellular basis of neurological disorders have advanced at a slow pace, especially due to the extreme invasiveness of brain biopsying and limitations of cell lines and animal models that have been used. Since the derivation of pluripotent stem cells (PSCs), a novel source of cells for regenerative medicine and disease modeling has become available, holding great potential for the neurology field. However, safety for therapy and accurateness for modeling have been a matter of intense debate, considering that genomic instability, including the gain and loss of chromosomes (aneuploidy), has been repeatedly observed in those cells. Despite the fact that recent reports have described some degree of aneuploidy as being normal during neuronal differentiation and present in healthy human brains, this phenomenon is particularly controversial since it has traditionally been associated with cancer and disabling syndromes. It is therefore necessary to appreciate, to which extent, aneuploid pluripotent stem cells are suitable for regenerative medicine and neurological modeling and also the limits that separate constitutive from disease-related aneuploidy. In this review, recent findings regarding chromosomal instability in PSCs and within the brain will be discussed.

STI1 promotes glioma proliferation through MAPK and PI3K pathways

Gliomas are tumors derived from glia or their precursors within the central nervous system. Clinically, gliomas are divided into four grades and the glioblastoma multiforme (GBM), also referred as grade IV astrocytoma, is the most aggressive and the most common glioma in humans. The prognosis for patients with GBM remains dismal, with a median survival of 9–12 months. Despite their striking heterogeneity, common alterations in specific cellular signal transduction pathways occur within most GBMs. Previous work from our group identified the co‐chaperone stress‐inducible protein 1 (STI1) as a cell surface ligand for cellular prion (PrPC), which leads to the activation of several signal transduction pathways, some of which modulate cell survival. In the present work, we used thymidine incorporation assays to investigate the effect of STI1 upon proliferation of the human glioblastoma‐derived cell line A172. Here we report that STI1 is secreted by and induces proliferation in tumor cells, an effect that is modulated by the Erk and PI3K pathways, and that, in contrast to glioma cells, STI1 does not induce proliferation of normal glia. In addition, our data suggest the involvement of PrPC in STI1‐induced proliferation of A172 cells. These results provide initial evidence of a new functional role for STI1 on the physiology of human gliomas, and may lead to the identification of new therapeutic targets in these tumors.

Activation of NMDA receptors protects against glutamate neurotoxicity in the retina: evidence for the involvement of neurotrophins

Activation of glutamate receptors has been implicated in excitotoxicity. Here, we have investigated whether subtoxic concentrations of glutamate can modulate neuronal death in the developing retina. Explants of rat retinas were pre-incubated with glutamate, N-methyl-d-aspartate (NMDA), kainate, quisqualate or trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) for 18 h. Then, glutamate (6 mM) was added to the explants for an additional 6 h. Glutamate-induced degeneration was restricted to the emerging inner nuclear layer. Pre-incubation with glutamate, NMDA, or both, reduced glutamate-induced neuronal death and protected against neuronal death induced by irradiation (2 Gy). The NMDA receptor antagonists, 2-amino-5-phosphonovaleric acid (d-APV; 30 microM) or 5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine hydrogen maleate (MK-801; 30 microM), prevented glutamate-induced neuroprotection. To investigate whether this neuroprotection was mediated by neurotrophins, we incubated retinal explants with either brain-derived neurotrophic factor or neurotrophin-4. Both treatments resulted in partial protection against glutamate-induced neurotoxicity. Furthermore, NMDA mediated neuroprotection was totally reversed when a soluble form of the specific tyrosine kinase receptor B was simultaneously added to the explants. Our results suggest that activation of NMDA receptors may control neuronal death in the retina during development. This modulation seems to depend, at least in part, on the release of neurotrophins within the retina.

Developmental sources of conservation and variation in the evolution of the primate eye

Conserved developmental programs, such as the order of neurogenesis in the mammalian eye, suggest the presence of useful features for evolutionary stability and variability. The owl monkey, Aotus azarae, has developed a fully nocturnal retina in recent evolution. Description and quantification of cell cycle kinetics show that embryonic cytogenesis is extended in Aotus compared with the diurnal New World monkey Cebus apella. Combined with the conserved mammalian pattern of retinal cell specification, this single change in retinal progenitor cell proliferation can produce the multiple alterations of the nocturnal retina, including coordinated reduction in cone and ganglion cell numbers, increase in rod and rod bipolar numbers, and potentially loss of the fovea.

N-myc coordinates retinal growth with eye size during mouse development

Myc family members play crucial roles in regulating cell proliferation, size, differentiation, and survival during development. We found that N-myc is expressed in retinal progenitor cells, where it regulates proliferation in a cell-autonomous manner. In addition, N-myc coordinates the growth of the retina and eye. Specifically, the retinas of N-myc-deficient mice are hypocellular but are precisely proportioned to the size of the eye. N-myc represses the expression of the cyclin-dependent kinase inhibitor p27Kip1 but acts independently of cyclin D1, the major D-type cyclin in the developing mouse retina. Acute inactivation of N-myc leads to increased expression of p27Kip1, and simultaneous inactivation of p27Kip1 and N-myc rescues the hypocellular phenotype in N-myc-deficient retinas. N-myc is not required for retinal cell fate specification, differentiation, or survival. These data represent the first example of a role for a Myc family member in retinal development and the first characterization of a mouse model in which the hypocellular retina is properly proportioned to the other ocular structures. We propose that N-myc lies upstream of the cell cycle machinery in the developing mouse retina and thus coordinates the growth of both the retina and eye through extrinsic cues.

Control of programmed cell death by neurotransmitters and neuropeptides in the developing mammalian retina

It has long been known that a barrage of signals from neighboring and connecting cells, as well as components of the extracellular matrix, control cell survival. Given the extensive repertoire of retinal neurotransmitters, neuromodulators and neurotrophic factors, and the exhuberant interconnectivity of retinal interneurons, it is likely that various classes of released neuroactive substances may be involved in the control of sensitivity to retinal cell death. The aim of this article is to review evidence that neurotransmitters and neuropeptides control the sensitivity to programmed cell death in the developing retina. Whereas the best understood mechanism of execution of cell death is that of caspase-mediated apoptosis, current evidence shows that not only there are many parallel pathways to apoptotic cell death, but non-apoptotic programs of execution of cell death are also available, and may be triggered either in isolation or combined with apoptosis. The experimental data show that many upstream signaling pathways can modulate cell death, including those dependent on the second messengers cAMP-PKA, calcium and nitric oxide. Evidence for anterograde neurotrophic control is provided by a variety of models of the central nervous system, and the data reviewed here indicate that an early function of certain neurotransmitters, such as glutamate and dopamine, as well as neuropeptides such as pituitary adenylyl cyclase-activating polypeptide and vasoactive intestinal peptide is the trophic support of cell populations in the developing retina. This may have implications both regarding the mechanisms of retinal organogenesis, as well as pathological conditions leading to retinal dystrophies and to dysfunctional cellular behavior.

Arginase-1 expression in granulomas of tuberculosis patients

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen able to survive and multiply within macrophages. Several mechanisms allow this bacterium to escape macrophage microbicidal activity. Mtb may interfere with the ability of mouse macrophages to produce antibactericidal nitric oxide, by inducing the expression of arginase 1 (Arg1). It remains unclear whether this pathway has a role in humans infected with Mtb. In this study, we investigated the expression of Arg1 in granulomas of human lung tissues from patients with tuberculosis. We show that Arg1 is expressed not only in granuloma-associated macrophages, but also in type II pneumocytes.

Pituitary adenylyl cyclase-activating polypeptide controls the proliferation of retinal progenitor cells through downregulation of cyclin D1.

During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase‐activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP–cAMP‐dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP‐specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [3H]thymidine as well as the number of 5‐bromo‐2′‐deoxyuridine‐positive and cyclin D1‐positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP–PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell‐extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells.

NMDA receptor activation modulates programmed cell death during early post-natal retinal development: a BDNF-dependent mechanism

Glutamate is a classical excitotoxin of the central nervous system (CNS), but extensive work demonstrates neuroprotective roles of this neurotransmitter in developing CNS. Mechanisms of glutamate‐mediated neuroprotection are still under scrutiny. In this study, we investigated mediators of glutamate‐induced neuroprotection, and tested whether this neurotransmitter controls programmed cell death in the developing retina. The protective effect of N‐methyl‐d‐aspartate (NMDA) upon differentiating cells of retinal explants was completely blocked by a neutralizing antibody to brain‐derived neurotrophic factor (BDNF), but not by an antibody to neurotrophin‐4 (NT‐4). Consistently, chronic activation of NMDA receptor increased the expression of BDNF and trkB mRNA, as well as BDNF protein content, but did not change the content of NT‐4 mRNA in retinal tissue. Furthermore, we showed that in vivo inactivation of NMDA receptor by intraperitoneal injections of MK‐801 increased natural cell death of specific cell populations of the post‐natal retina. Our results show that chronic activation of NMDA receptors in vitro induces a BDNF‐dependent neuroprotective state in differentiating retinal cells, and that NMDA receptor activation controls programmed cell death of developing retinal neurons in vivo.

Neuroimmunoendocrine Regulation of the Prion Protein in Neutrophils

Background: Prion protein (PrPC) modulates inflammation, and prion diseases affect neutrophil numbers and functions, but the regulation of PrPC in neutrophils is unknown. Results: Inflammation and stress massively up-regulated PrPC in neutrophils via glucocorticoids and TGF-β. Conclusion: We show a novel pathway of regulation of PrPC, with functional consequences for neutrophils. Significance: Systemic control of the expression and function of PrPC broadly modulates cellular physiology and pathology. The prion protein (PrPC) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrPC may be implicated in other degenerative conditions often associated with inflammation. PrPC is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrPC in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrPC in mouse neutrophils. Up-regulation of PrPC was dependent on the serum content of TGF-β and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-β, either alone or in combination, directly up-regulated PrPC in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrPC. Moreover, GC also mediated up-regulation of PrPC in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrPC presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrPC in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems.