Seed‐to‐Seedling Transitions Exhibit Distance‐Dependent Mortality but No Strong Spacing Effects in a Neotropical Forest

Patterns of seed dispersal and seed mortality influence the spatial structure of plant communities and the local coexistence of competing species. Most seeds are dispersed in proximity to the parent tree, where mortality is also expected to be the highest, because of competition with siblings or the attraction of natural enemies. Whereas distance‐dependent mortality in the seed‐to‐seedling transition was often observed in tropical forests, few studies have attempted to estimate the shape of the survival‐distance curves, which determines whether the peak of seedling establishment occurs away from the parent tree (Janzen–Connell pattern) or if the peak attenuates but remains at the parent location (Hubbell pattern). In this study, we inferred the probability density of seed dispersal and two stages of seedling establishment (new recruits, and seedlings 20 cm or taller) with distance for 24 tree species present in the 50‐ha Forest Dynamics Plot of Barro Colorado Island, Panama. Using data from seed traps, seedling survey quadrats, and tree‐census records spanning the 1988–2014 period, we fit hierarchical Bayesian models including parameters for tree fecundity, the shape of the dispersal kernel, and overdispersion of seed or seedling counts. We combined predictions from multiple dispersal kernels to obtain more robust inferences. We find that Hubbell patterns are the most common and Janzen–Connell patterns are very rare among those species; that distance‐dependent mortality may be stronger in the seed stage, in the early recruit stage, or comparable in both; and that species with larger seeds experience less overall mortality and less distance‐dependent mortality. Finally, we describe how this modeling approach could be extended at a community scale to include less abundant species

Emergence of Microglia Bearing Senescence Markers During Paralysis Progression in a Rat Model of Inherited ALS

Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1G93A mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1G93A rats, numerous cells displayed nuclear p16INK4a as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16INK4a-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16INK4a-positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1G93A rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days in vitro and subsequent passages. Senescent SOD1G93A microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-β-Gal) activity as well as positive labeling for p16INK4a, p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16INK4a staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1G93A rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS

Nitric oxide-mediated oxidative damage and the progressive demise of motor neurons in ALS

Oxidative damage is a common and early feature of Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Dr. Mark Smith and his colleagues have built the case for oxidative stress being a primary progenitor rather than a secondary end-stage epiphenomenon of neurodegeneration. They proposed that reactive oxygen species contribute to the “age-related cascade of neurodegeneration” whereby accumulative oxidative damage with age promotes other characteristic pathological changes in afflicted brain regions, including protein aggregation, metabolic deficiencies, and inflammation. Nitric oxide (NO) likely plays a critical role in this age-related cascade. NO is a major signaling molecule produced in the central nervous system (CNS) to modulate neurological activity through stimulating cyclic GMP synthesis. However, the same physiological concentrations of NO relevant in cellular signaling may also initiate and amplify oxidative damage by diffusion-limited reactions with superoxide (O₂[superscript •−]) to produce peroxynitrite (ONOO⁻). This is perhaps best illustrated in ALS where physiological levels of NO promote survival of motor neurons, but the same concentrations can stimulate motor neuron apoptosis and glial cell activation under pathological conditions. While these changes represent a complex mechanism involving multiple cell types in the pathogenesis of ALS, they also reveal general processes underlying neurodegeneration.Keywords: peroxynitrite, protein nitration, amyotrophic lateral sclerosis, nitric oxide, neurodegenerationKeywords: peroxynitrite, protein nitration, amyotrophic lateral sclerosis, nitric oxide, neurodegeneratio

Electron Capture, Collision-Induced, and Electron Capture-Collision Induced Dissociation in Q-TOF

Recently, we demonstrated that a radio-frequency-free electromagnetostatic (RF-free EMS) cell could be retrofitted into a triple quad mass spectrometer to allow electron-capture dissociation (ECD) without the aid of cooling gas or phase-specific electron injection into the cell [1-2]. Subsequently, we used our RF-free EMS cell in the same instrument platform to demonstrate ECD occurring in the same space and at the same time with collision-induced dissociation (CID) to produce golden pairs and even triplets from peptides [3]. In this report, we demonstrate that ECD and CID product-ion mass spectra can be recorded at high resolution with flexible control of fragmentation processes using a newly designed cell installed in a hybrid Q-TOF tandem mass spectrometer.This is an author's manuscript. The published article is copyrighted by Springer and can be found at: https://doi.org/10.1007/s13361-010-0072-xKeywords: A radio-frequency-free electromagnetostatic cell, Electron capture dissociationKeywords: A radio-frequency-free electromagnetostatic cell, Electron capture dissociatio

ECD of Tyrosine Phosphorylation in a Triple Quadrupole Mass Spectrometer with a Radio-Frequency-Free Electromagnetostatic Cell

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell

Post-paralysis tyrosine kinase inhibition with masitinib abrogates neuroinflammation and slows disease progression in inherited amyotrophic lateral sclerosis

Background: In the SOD1G93A mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS. Methods: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1G93A rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset. Results: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1G93A spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1G93A rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %. Conclusions: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.Agencia Nacional de Investigación e Innovació

Emergence of Microglia Bearing Senescence Markers During Paralysis Progression in a Rat Model of Inherited ALS

Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1(G93A) mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1(G93A) rats, numerous cells displayed nuclear p16(INK4a) as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16(INK4a)-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16(INK4a)-positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1(G93A) rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days in vitro and subsequent passages. Senescent SOD1(G93A) microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-beta-Gal) activity as well as positive labeling for p16(INK4a), p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16(INK4a) staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1(G93A) rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS

Nitration of Hsp90 induces cell death

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies

Phenotypic transition of microglia into astrocyte-like cells associated with disease onset in a model of inherited ALS

Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the Amyotrophic lateral sclerosis (ALS)-linked SOD1ᴳ⁹³ᴬ mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of prolifer- ative astrocyte-like cells that surround motor neurons. These cells, designated as Aberrant Astrocytes (AbA cells) because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular origin of AbA cells remains unknown. Because AbA cells are labeled with the proliferation marker Ki67, we analyzed the phenotypic makers of proliferating glial cells that surround motor neurons by immunohistochemistry. The number of Ki67⁺AbA cells sharply increased in symptomatic rats, displaying large cell bodies with processes embracing motor neurons. Most were co-labeled with astrocytic marker GFAP concurrently with the microglial markers Iba1 and CD163. Cultures of spinal cord prepared from symptomatic SOD1ᴳ⁹³ᴬ rats yielded large numbers of microglia expressing Iba1, CD11b, and CD68. Cells sorted for CD11b expression by flow cytometry transformed into AbA cells within two weeks. During these two weeks, the expression of microglial markers largely disappeared, while GFAP and S100β expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1ᴳ⁹³ᴬ rats that undergo a phenotypic transition into AbA cells after onset of paralysis that may promote the fulminant disease progression. These cells could be a therapeutic target for slowing paralysis progression in ALS