Neonicotinoid Pesticides Affect Developing Neurons in Experimental Mouse Models and in Human Induced Pluripotent Stem Cell (iPSC)-Derived Neural Cultures and Organoids.

Neonicotinoids are synthetic, nicotine-derived insecticides used worldwide to protect crops and domestic animals from pest insects. The reported evidence shows that they are also able to interact with mammalian nicotine receptors (nAChRs), triggering detrimental responses in cultured neurons. Exposure to high neonicotinoid levels during the fetal period induces neurotoxicity in animal models. Considering the persistent exposure to these insecticides and the key role of nAChRs in brain development, their potential neurotoxicity on mammal central nervous system (CNS) needs further investigations. We studied here the neurodevelopmental effects of different generations of neonicotinoids on CNS cells in mouse fetal brain and primary cultures and in neuronal cells and organoids obtained from human induced pluripotent stem cells (iPSC). Neonicotinoids significantly affect neuron viability, with imidacloprid (IMI) inducing relevant alterations in synaptic protein expression, neurofilament structures, and microglia activation in vitro, and in the brain of prenatally exposed mouse fetuses. IMI induces neurotoxic effects also on developing human iPSC-derived neurons and cortical organoids. Collectively, the current findings show that neonicotinoids might induce impairment during neuro/immune-development in mouse and human CNS cells and provide new insights in the characterization of risk for the exposure to this class of pesticides.

SARS-CoV-2 Nucleocapsid Protein Induces Tau Pathological Changes That Can Be Counteracted by SUMO2.

Neurologic manifestations are an immediate consequence of SARS-CoV-2 infection, the etiologic agent of COVID-19, which, however, may also trigger long-term neurological effects. Notably, COVID-19 patients with neurological symptoms show elevated levels of biomarkers associated with brain injury, including Tau proteins linked to Alzheimer's pathology. Studies in brain organoids revealed that SARS-CoV-2 alters the phosphorylation and distribution of Tau in infected neurons, but the mechanisms are currently unknown. We hypothesize that these pathological changes are due to the recruitment of Tau into stress granules (SGs) operated by the nucleocapsid protein (NCAP) of SARS-CoV-2. To test this hypothesis, we investigated whether NCAP interacts with Tau and localizes to SGs in hippocampal neurons in vitro and in vivo. Mechanistically, we tested whether SUMOylation, a posttranslational modification of NCAP and Tau, modulates their distribution in SGs and their pathological interaction. We found that NCAP and Tau colocalize and physically interact. We also found that NCAP induces hyperphosphorylation of Tau and causes cognitive impairment in mice infected with NCAP in their hippocampus. Finally, we found that SUMOylation modulates NCAP SG formation in vitro and cognitive performance in infected mice. Our data demonstrate that NCAP induces Tau pathological changes both in vitro and in vivo. Moreover, we demonstrate that SUMO2 ameliorates NCAP-induced Tau pathology, highlighting the importance of the SUMOylation pathway as a target of intervention against neurotoxic insults, such as Tau oligomers and viral infection.

Pseudo-Orthostatic Tremor in Graves' Disease: A Possible Early Sign of Parkinsonism?

Background: Pseudo-orthostatic tremor is a hyperkinetic movement disorder usually associated with other neurological comorbidities, mainly Parkinson's disease. Case report: A 65-year-old male presented with unsteadiness and leg tremor while standing. Electrophysiological evaluation confirmed the presence of pseudo-orthostatic tremor. Blood test showed an undiagnosed Graves' disease. A complete remission of tremor was achieved with methimazole. Dopamine transporter scintigraphy showed a mild reduction of the striatal binding, bilaterally. Discussion: Graves' disease can be associated with pseudo-orthostatic tremor. Thyroid function should be assessed in patients complaining of unsteadiness. The causative role of hyperthyroidism in determining dopaminergic degeneration and uncovering subclinical parkinsonism warrants further investigations.

Synergistic Pharmacological Therapy to Modulate Glial Cells in Spinal Cord Injury.

Current treatments for modulating the glial-mediated inflammatory response after spinal cord injury (SCI) have limited ability to improve recovery. This is quite likely due to the lack of a selective therapeutic approach acting on microgliosis and astrocytosis, the glia components most involved after trauma, while maximizing efficacy and minimizing side effects. A new nanogel that can selectively release active compounds in microglial cells and astrocytes is developed and characterized. The degree of selectivity and subcellular distribution of the nanogel is evaluated by applying an innovative super-resolution microscopy technique, expansion microscopy. Two different administration schemes are then tested in a SCI mouse model: in an early phase, the nanogel loaded with Rolipram, an anti-inflammatory drug, achieves significant improvement in the animal's motor performance due to the increased recruitment of microglia and macrophages that are able to localize the lesion. Treatment in the late phase, however, gives opposite results, with worse motor recovery because of the widespread degeneration. These findings demonstrate that the nanovector can be selective and functional in the treatment of the glial component in different phases of SCI. They also open a new therapeutic scenario for tackling glia-mediated inflammation after neurodegenerative events in the central nervous system.

Effect of Lactoferrin on Clinical Outcomes of Hospitalized Patients with COVID-19: The LAC Randomized Clinical Trial.

As lactoferrin is a nutritional supplement with proven antiviral and immunomodulatory abilities, it may be used to improve the clinical course of COVID-19. The clinical efficacy and safety of bovine lactoferrin were evaluated in the LAC randomized double-blind placebo-controlled trial. A total of 218 hospitalized adult patients with moderate-to-severe COVID-19 were randomized to receive 800 mg/die oral bovine lactoferrin (n = 113) or placebo (n = 105), both given in combination with standard COVID-19 therapy. No differences in lactoferrin vs. placebo were observed in the primary outcomes: the proportion of death or intensive care unit admission (risk ratio of 1.06 (95% CI 0.63-1.79)) or proportion of discharge or National Early Warning Score 2 (NEWS2) ≤ 2 within 14 days from enrollment (RR of 0.85 (95% CI 0.70-1.04)). Lactoferrin showed an excellent safety and tolerability profile. Even though bovine lactoferrin is safe and tolerable, our results do not support its use in hospitalized patients with moderate-to-severe COVID-19.

Neural cortical organoids from self-assembling human iPSC as a model to investigate neurotoxicity in brain ischemia.

Brain ischemia is a common acute injury resulting from impaired blood flow to the brain. Translation of effective drug candidates from experimental models to patients has systematically failed. The use of human induced pluripotent stem cells (iPSC) offers new opportunities to gain translational insights into diseases including brain ischemia. We used a human 3D self-assembling iPSC-derived model (human cortical organoids, hCO) to characterize the effects of ischemia caused by oxygen-glucose deprivation (OGD). hCO exposed to 2 h or 8 h of OGD had neuronal death and impaired neuronal network complexity, measured in whole-mounting microtubule-associated protein 2 (MAP-2) immunostaining. Neuronal vulnerability was reflected by a reduction in MAP-2 mRNA levels, and increased release of neurofilament light chain (NfL) in culture media, proportional to OGD severity. Glial fibrillary acidic protein (GFAP) gene or protein levels did not change in hCO, but their release in medium increased after prolonged OGD. In conclusion, this human 3D iPSC-based in vitro model of brain ischemic injury is characterized by marked neuronal injury reflected by the release of the translational biomarker NfL which is relevant for testing neuroprotective strategies.

An integrated approach, based on mass spectrometry, for the assessment of imidacloprid metabolism and penetration into mouse brain and fetus after oral treatment.

Imidacloprid is an insecticide belonging to neonicotinoids, a class of agonists of the nicotinic acetylcholine receptors that shows higher affinities in insects compared to mammals. However, recent evidence show that neonicotinoids can bind to the mammalian receptors, leading to detrimental responses in cultured neurons. We developed an analytical strategy which uses mass spectrometry with multiple reaction monitoring (targeted approach) and high-resolution acquisitions (untargeted approach), which were applied to quantify imidacloprid and to identify its metabolites in biological tissues after oral treatments of mice. Mouse dams were treated with doses from 0.118 mg/kg bw day up to 41 mg/kg day between gestational days 6-9. Results showed quantifiable levels of imidacloprid in plasma (from 30.48 to 5705 ng/mL) and brain (from 20.48 to 5852 ng/g) of treated mice, proving the passage through the mammalian blood-brain barrier with a high correspondence between doses and measured concentrations. Untargeted analyses allowed the identification of eight metabolites including imidacloprid-olefin, hydroxy-imidacloprid dihydroxy-imidacloprid, imidacloprid-nitrosimine, desnitro-imidacloprid, 6-chloronicotinic acid, 5-(methylsulfanyl)pyridine-2-carboxylic acid and N-imidazolidin-2-ylidenenitramide in plasma and brain. Moreover, analysis of embryonic tissues after oral treatment of mouse dams showed detectable levels of imidacloprid (816.6 ng/g after a dose of 4.1 mg/Kg bw day and 5646 ng/g after a dose of 41 mg/Kg bw day) and its metabolites, proving the permeability of the placenta barrier. Although many studies have been reported on the neurotoxicity of neonicotinoids, our study paves the way for a risk assessment in neurodevelopmental toxicity, demostrating the capability of imidacloprid and its metabolites to pass the biological barriers.