Pathogenic or protective? Neuropeptide Y in amyotrophic lateral sclerosis.

Neuropeptide Y (NPY) is an endogenous peptide of the central and enteric nervous systems which has gained significant interest as a potential neuroprotective agent for treatment of neurodegenerative disease. Amyotrophic lateral sclerosis (ALS) is an aggressive and fatal neurodegenerative disease characterized by motor deficits and motor neuron loss. In ALS, recent evidence from ALS patients and animal models has indicated that NPY may have a role in the disease pathogenesis. Increased NPY levels were found to correlate with disease progression in ALS patients. Similarly, NPY expression is increased in the motor cortex of ALS mice by end stages of the disease. Although the functional consequence of increased NPY levels in ALS is currently unknown, NPY has been shown to exert a diverse range of neuroprotective roles in other neurodegenerative diseases; through modulation of potassium channel activity, increased production of neurotrophins, inhibition of endoplasmic reticulum stress and autophagy, reduction of excitotoxicity, oxidative stress, neuroinflammation and hyperexcitability. Several of these mechanisms and signalling pathways are heavily implicated in the pathogenesis of ALS. Therefore, in this review, we discuss possible effects of NPY and NPY-receptor signalling in the ALS disease context, as determining NPY's contribution to, or impact on, ALS disease mechanisms will be essential for future studies investigating the NPY system as a therapeutic strategy in this devastating disease.

Biological nitrogen fixation in maize: optimizing nitrogenase expression in a root-associated diazotroph.

Plants depend upon beneficial interactions between roots and root-associated microorganisms for growth promotion, disease suppression, and nutrient availability. This includes the ability of free-living diazotrophic bacteria to supply nitrogen, an ecological role that has been long underappreciated in modern agriculture for efficient crop production systems. Long-term ecological studies in legume-rhizobia interactions have shown that elevated nitrogen inputs can lead to the evolution of less cooperative nitrogen-fixing mutualists. Here we describe how reprogramming the genetic regulation of nitrogen fixation and assimilation in a novel root-associated diazotroph can restore ammonium production in the presence of exogenous nitrogen inputs. We isolated a strain of the plant-associated proteobacterium Kosakonia sacchari from corn roots, characterized its nitrogen regulatory network, and targeted key nodes for gene editing to optimize nitrogen fixation in corn. While the wild-type strain exhibits repression of nitrogen fixation in conditions replete with bioavailable nitrogen, such as fertilized greenhouse and field experiments, remodeled strains show elevated levels in the rhizosphere of corn in the greenhouse and field even in the presence of exogenous nitrogen. Such strains could be used in commercial applications to supply fixed nitrogen to cereal crops.

Synapse Dysfunction of Layer V Pyramidal Neurons Precedes Neurodegeneration in a Mouse Model of TDP-43 Proteinopathies.

TDP-43 is a major protein component of pathological neuronal inclusions that are present in frontotemporal dementia and amyotrophic lateral sclerosis. We report that TDP-43 plays an important role in dendritic spine formation in the cortex. The density of spines on YFP+ pyramidal neurons in both the motor and somatosensory cortex of Thy1-YFP mice, increased significantly from postnatal day 30 (P30), to peak at P60, before being pruned by P90. By comparison, dendritic spine density was significantly reduced in the motor cortex of Thy1-YFP::TDP-43A315T transgenic mice prior to symptom onset (P60), and in the motor and somatosensory cortex at symptom onset (P90). Morphological spine-type analysis revealed that there was a significant impairment in the development of basal mushroom spines in the motor cortex of Thy1-YFP::TDP-43A315T mice compared to Thy1-YFP control. Furthermore, reductions in spine density corresponded to mislocalisation of TDP-43 immunoreactivity and lowered efficacy of synaptic transmission as determined by electrophysiology at P60. We conclude that mutated TDP-43 has a significant pathological effect at the dendritic spine that is associated with attenuated neural transmission.

Amyloid ß precursor protein regulates neuron survival and maturation in the adult mouse brain.

The amyloid-ß precursor protein (APP) is a transmembrane protein that is widely expressed within the central nervous system (CNS). While the pathogenic dysfunction of this protein has been extensively studied in the context of Alzheimer's disease, its normal function is poorly understood, and reports have often appeared contradictory. In this study we have examined the role of APP in regulating neurogenesis in the adult mouse brain by comparing neural stem cell proliferation, as well as new neuron number and morphology between APP knockout mice and C57bl6 controls. Short-term EdU administration revealed that the number of proliferating EdU+ neural progenitor cells and the number of PSA-NCAM+ neuroblasts produced in the SVZ and dentate gyrus were not affected by the life-long absence of APP. However, by labelling newborn cells with EdU and then following their fate over-time, we determined that ~48% more newly generated EdU+ NeuN+ neurons accumulated in the granule cell layer of the olfactory bulb and ~57% more in the dentate gyrus of young adult APP knockout mice relative to C57bl6 controls. Furthermore, proportionally fewer of the adult-born olfactory bulb granule neurons were calretinin+. To determine whether APP was having an effect on neuronal maturation, we administered tamoxifen to young adult Nestin-CreERT2::Rosa26-YFP and Nestin-CreERT2::Rosa26-YFP::APP-knockout mice, fluorescently labelling ~80% of newborn (EdU+) NeuN+ dentate granule neurons formed between P75 and P105. Our analysis of their morphology revealed that neurons added to the hippocampus of APP knockout mice have shorter dendritic arbors and only half the number of branch points as those generated in C57bl6 mice. We conclude that APP reduces the survival of newborn neurons in the olfactory bulb and hippocampus, but that it does not influence all neuronal subtypes equally. Additionally, APP influences dentate granule neuron maturation, acting as a robust regulator of dendritic extension and arborisation.

Synaptic remodeling follows upper motor neuron hyperexcitability in a rodent model of TDP-43.

Amyotrophic Lateral Sclerosis (ALS) is an incurable disease characterized by relentlessly progressive degeneration of the corticomotor system. Cortical hyperexcitability has been identified as an early pre-symptomatic biomarker of ALS. This suggests that hyperexcitability occurs upstream in the ALS pathological cascade and may even be part of the mechanism that drives development of symptoms or loss of motor neurons in the spinal cord. However, many studies also indicate a loss to the synaptic machinery that mediates synaptic input which raises the question of which is the driver of disease, and which is a homeostatic response. Herein, we used an inducible mouse model of TDP-43 mediated ALS that permits for the construction of detailed phenotypic timelines. Our work comprehensively describes the relationship between intrinsic hyperexcitability and altered synaptic input onto motor cortical layer 5 pyramidal neurons over time. As a result, we have constructed the most complete timeline of electrophysiological changes following induction of TDP-43 dysfunction in the motor cortex. We report that intrinsic hyperexcitability of layer 5 pyramidal neurons precedes changes to excitatory synaptic connections, which manifest as an overall loss of inputs onto layer 5 pyramidal neurons. This finding highlights the importance of hyperexcitability as a primary mechanism of ALS and re-contextualizes synaptic changes as possibly representing secondary adaptive responses. Recognition of the relationship between intrinsic hyperexcitability and reduced excitatory synaptic input has important implications for the development of useful therapies against ALS. Novel strategies will need to be developed that target neuronal output by managing excitability against synapses separately.

Intranasal neuropeptide Y1 receptor antagonism improves motor deficits in symptomatic SOD1 ALS mice.

OBJECTIVE: Neuropeptide Y (NPY) is a 36 amino acid peptide widely considered to provide neuroprotection in a range of neurodegenerative diseases. In the fatal motor neuron disease amyotrophic lateral sclerosis (ALS), recent evidence supports a link between NPY and ALS disease processes. The goal of this study was to determine the therapeutic potential and role of NPY in ALS, harnessing the brain-targeted intranasal delivery of the peptide, previously utilised to correct motor and cognitive phenotypes in other neurological conditions. METHODS: To confirm the association with clinical disease characteristics, NPY expression was quantified in post-mortem motor cortex tissue of ALS patients and age-matched controls. The effect of NPY on ALS cortical pathophysiology was investigated using slice electrophysiology and multi-electrode array recordings of SOD1G93A cortical cultures in vitro. The impact of NPY on ALS disease trajectory was investigated by treating SOD1G93A mice intranasally with NPY and selective NPY receptor agonists and antagonists from pre-symptomatic and symptomatic phases of disease. RESULTS: In the human post-mortem ALS motor cortex, we observe a significant increase in NPY expression, which is not present in the somatosensory cortex. In vitro, we demonstrate that NPY can ameliorate ALS hyperexcitability, while brain-targeted nasal delivery of NPY and a selective NPY Y1 receptor antagonist modified survival and motor deficits specifically within the symptomatic phase of the disease in the ALS SOD1G93A mouse. INTERPRETATION: Taken together, these findings highlight the capacity for non-invasive brain-targeted interventions in ALS and support antagonism of NPY Y1Rs as a novel strategy to improve ALS motor function.

Differential NPY-Y1 Receptor Density in the Motor Cortex of ALS Patients and Familial Model of ALS.

Destabilization of faciliatory and inhibitory circuits is an important feature of corticomotor pathology in amyotrophic lateral sclerosis (ALS). While GABAergic inputs to upper motor neurons are reduced in models of the disease, less understood is the involvement of peptidergic inputs to upper motor neurons in ALS. The neuropeptide Y (NPY) system has been shown to confer neuroprotection against numerous pathogenic mechanisms implicated in ALS. However, little is known about how the NPY system functions in the motor system. Herein, we investigate post-synaptic NPY signaling on upper motor neurons in the rodent and human motor cortex, and on cortical neuron populations in vitro. Using immunohistochemistry, we show the increased density of NPY-Y1 receptors on the soma of SMI32-positive upper motor neurons in post-mortem ALS cases and SOD1G93A excitatory cortical neurons in vitro. Analysis of receptor density on Thy1-YFP-H-positive upper motor neurons in wild-type and SOD1G93A mouse tissue revealed that the distribution of NPY-Y1 receptors was changed on the apical processes at early-symptomatic and late-symptomatic disease stages. Together, our data demonstrate the differential density of NPY-Y1 receptors on upper motor neurons in a familial model of ALS and in ALS cases, indicating a novel pathway that may be targeted to modulate upper motor neuron activity.

Enabling Biological Nitrogen Fixation for Cereal Crops in Fertilized Fields.

Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 ("Kv137-1036") retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

Amyotrophic lateral sclerosis mutant TDP-43 may cause synaptic dysfunction through altered dendritic spine function.

Altered cortical excitability and synapse dysfunction are early pathogenic events in amyotrophic lateral sclerosis (ALS) patients and animal models. Recent studies propose an important role for TAR DNA-binding protein 43 (TDP-43), the mislocalization and aggregation of which are key pathological features of ALS. However, the relationship between ALS-linked TDP-43 mutations, excitability and synaptic function is not fully understood. Here, we investigate the role of ALS-linked mutant TDP-43 in synapse formation by examining the morphological, immunocytochemical and excitability profile of transgenic mouse primary cortical pyramidal neurons that over-express human TDP-43A315T In TDP-43A315T cortical neurons, dendritic spine density was significantly reduced compared to wild-type controls. TDP-43A315T over-expression increased the total levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropinionic acid (AMPA) glutamate receptor subunit GluR1, yet the localization of GluR1 to the dendritic spine was reduced. These postsynaptic changes were coupled with a decrease in the amount of the presynaptic marker synaptophysin that colocalized with dendritic spines. Interestingly, action potential generation was reduced in TDP-43A315T pyramidal neurons. This work reveals a crucial effect of the over-expression mutation TDP-43A315T on the formation of synaptic structures and the recruitment of GluR1 to the synaptic membrane. This pathogenic effect may be mediated by cytoplasmic mislocalization of TDP-43A315T Loss of synaptic GluR1, and reduced excitability within pyramidal neurons, implicates hypoexcitability and attenuated synaptic function in the pathogenic decline of neuronal function in TDP-43-associated ALS. Further studies into the mechanisms underlying AMPA receptor-mediated excitability changes within the ALS cortical circuitry may yield novel therapeutic targets for treatment of this devastating disease.

Reduced Excitability and Increased Neurite Complexity of Cortical Interneurons in a Familial Mouse Model of Amyotrophic Lateral Sclerosis.

Cortical interneurons play a crucial role in regulating inhibitory-excitatory balance in brain circuits, filtering synaptic information and dictating the activity of pyramidal cells through the release of GABA. In the fatal motor neuron (MN) disease, amyotrophic lateral sclerosis (ALS), an imbalance between excitation and inhibition is an early event in the motor cortex, preceding the development of overt clinical symptoms. Patients with both sporadic and familial forms of the disease exhibit reduced cortical inhibition, including patients with mutations in the copper/zinc superoxide-dismutase-1 (SOD1) gene. In this study, we investigated the influence of the familial disease-causing hSOD1-G93A ALS mutation on cortical interneurons in neuronal networks. We performed whole-cell patch-clamp recordings and neurobiotin tracing from GFP positive interneurons in primary cortical cultures derived from Gad67-GFP::hSOD1G93A mouse embryos. Targeted recordings revealed no overt differences in the passive properties of Gad67-GFP::hSOD1G93A interneurons, however the peak outward current was significantly diminished and cells were less excitable compared to Gad67-GFP::WT controls. Post hoc neurite reconstruction identified a significantly increased morphological complexity of the Gad67-GFP::hSOD1G93A interneuron neurite arbor compared to Gad67-GFP::WT controls. Our results from the SOD1 model suggest that cortical interneurons have electrophysiological and morphological alterations that could contribute to attenuated inhibitory function in the disease. Determining if these phenomena are driven by the network or represent intrinsic alteration of the interneuron may help explain the emergence of inhibitory susceptibility and ultimately disrupted excitability, in ALS.

Calretinin and Neuropeptide Y interneurons are differentially altered in the motor cortex of the SOD1G93A mouse model of ALS.

Increasing evidence indicates an excitatory/inhibitory imbalance may have a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Impaired inhibitory circuitry is consistently reported in the motor cortex of both familial and sporadic patients, closely associated with cortical hyperexcitability and ALS onset. Inhibitory network dysfunction is presumably mediated by intra-cortical inhibitory interneurons, however, the exact cell types responsible are yet to be identified. In this study we demonstrate dynamic changes in the number of calretinin- (CR) and neuropeptide Y-expressing (NPY) interneurons in the motor cortex of the familial hSOD1G93A ALS mouse model, suggesting their potential involvement in motor neuron circuitry defects. We show that the density of NPY-populations is significantly decreased by ~17% at symptom onset (8 weeks), and by end-stage disease (20 weeks) is significantly increased by ~30%. Conversely, the density of CR-populations is progressively reduced during later symptomatic stages (~31%) to end-stage (~36%), while CR-expressing interneurons also show alteration of neurite branching patterns at symptom onset. We conclude that a differential capacity for interneurons exists in the ALS motor cortex, which may not be a static phenomenon, but involves early dynamic changes throughout disease, implicating specific inhibitory circuitry.

Inhibitory dysfunction in amyotrophic lateral sclerosis: future therapeutic opportunities.

In amyotrophic lateral sclerosis, motor neuron hyperexcitability and inhibitory dysfunction is emerging as a potential causative link in the dysfunction and degeneration of the motoneuronal circuitry that characterizes the disease. Interneurons, as key regulators of excitability, may mediate much of this imbalance, yet we know little about the way in which inhibitory deficits perturb excitability. In this review, we explore inhibitory control of excitability and the potential contribution of altered inhibition to amyotrophic lateral sclerosis disease processes and vulnerabilities, identifying important windows of therapeutic opportunity and potential interventions, specifically targeting inhibitory control at key disease stages.

Event-related potentials reveal modelling of auditory repetition in the brain.

Two auditory event-related potential (ERP) waveforms, mismatch negativity (MMN) and repetition positivity (RP), are sensitive to repetition of auditory stimuli. Increasing repetition of standards produces larger MMN amplitudes to deviant stimuli in an oddball paradigm, known as the memory trace effect, and attributed to increasing strength of the memory trace for standards. RP to standards also increases as a function of repetition in a 'roving' oddball paradigm where the standard changes in pitch following presentation of a deviant tone. As the sensory memory trace representing standard stimuli must be continually updated in the roving paradigm, RP has been proposed to reflect memory trace formation. Given that RP to date has only been observed in roving oddball paradigms, we examined whether RP and the MMN memory trace effect are present in both roving and standard oddball paradigms in 24 young adults (mean age: 22.4±5years). Four, 8, or 16 standards preceded a deviant. We observed RP at Fz in standard ERPs in the roving but not constant paradigm. At mastoid sites, RP was observed in both paradigms. A memory trace effect was not observed at Fz in either paradigm. Our findings suggest that different generator sites in the brain model local and global auditory information with generators of mastoid activity primarily sensitive to local or short term stimulus history of auditory regularities while generators of frontal site activity retain more global information regarding stimulus history over a longer time period.

Efficacy of a scheduled IV cocktail of antiemetics for the palliation of nausea and vomiting in a hospice population.

This is a retrospective analysis of 10 mg metoclopramide, 25 mg diphenhydramine, and 4 mg dexamethasone given intravenous piggyback every 6 hours for nausea or vomiting. Outcome measures were rapidity of symptom relief based on the self-report of the patient and nursing documentation of relief from symptoms of nausea or vomiting. Seven hundred and ninety seven patients were admitted to the inpatient hospice unit during a 2-year period. Sixty-three patients developed nausea or vomiting requiring the cocktail. Fifty-seven patients (90%) had objective response as reflected in nursing notes. Symptom relief was usually noted within 2 days with improvement in oral intake and enjoyment in activities, such as parties and family interactions. Partial relief was noted in patients with gastrointestinal malignancies and peritoneal carcinomatosis even with the addition of other antiemetics to the cocktail.