Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs), however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout – Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.

Poly (ADP-ribose) Interacts With Phosphorylated α-Synuclein in Post Mortem PD Samples

Poly (ADP-ribose) (PAR) is a negatively charged polymer that is biosynthesized by Poly (ADP-ribose) Polymerase-1 (PARP-1) and regulates various cellular processes. Alpha-synuclein (αSyn) is an intrinsically disordered protein (IDP) that has been directly implicated with driving the onset and progression of Parkinson’s disease (PD). The mechanisms by which α-synuclein (αSyn) elicits its neurotoxic effects remain unclear, though it is well established that the main components of Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients are aggregated hyperphosphorylated (S129) forms of αSyn (pαSyn). In the present study, we used immunofluorescence-based assays to explore if PARP-1 enzymatic product (PAR) promotes the aberrant cytoplasmic accumulation of pαSyn. We also performed quantitative measurements using in situ proximity ligation assays (PLA) on a transgenic murine model of α-synucleinopathy (M83-SNCA∗A53T) and post mortem PD/PDD patient samples to characterize PAR–pαSyn interactions. Additionally, we used bioinformatic approaches and site-directed mutagenesis to identify PAR-binding regions on αSyn. In summary, our studies show that PAR–pαSyn interactions are predominantly observed in PD-relevant transgenic murine models of αSyn pathology and post mortem PD/PDD patient samples. Moreover, we confirm that the interactions between PAR and αSyn involve electrostatic forces between negatively charged PAR and lysine residues on the N-terminal region of αSyn.

Brain and Lung Cross-Protection against Ancestral or Emerging SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Murine Model

COVID-19 vaccines already in use or in clinical development may have safety concerns, limited immunogenicity in high-risk groups or reduced efficacy against emerging SARS-CoV-2 variants. In addition, although the neurotropism of SARS-CoV-2 is well established, the vaccine strategies currently developed have not taken into account the protection of the central nervous system. Here, we generated a transgenic mouse strain expressing the human Angiotensin Converting Enzyme 2, with unprecedented brain as well as lung permissibility to SARS-CoV-2 replication. Using this stringent transgenic model, we demonstrated that a non-integrative lentiviral vector, encoding for the spike glycoprotein of the ancestral Wuhan SARS-CoV-2, used in intramuscular prime and intranasal boost elicits sterilizing protection of lung and brain against both the Wuhan and the most genetically distant Manaus P.1 SARS-CoV-2 variants. Beyond the induction of strong neutralizing antibodies, the mechanism underlying this broad protection spectrum involves a robust protective spike-specific CD8+ T-cell immunity, unaffected by the recent mutations accumulated in the emerging SARS-CoV-2 variants.

BAMBI and CHGA in Prion Diseases: Neuropathological Assessment and Potential Role as Disease Biomarkers

Prion diseases affect both animals and humans. Research in the natural animal model of the disease could help in the understanding of neuropathological mechanisms and in the development of biomarkers for human pathologies. For this purpose, we studied the expression of 10 genes involved in prion propagation in vitro in the central nervous system of scrapie-infected sheep. Dysregulated genes (BAMBI and CHGA) were further analysed in a transgenic murine model (Tg338) of scrapie, and their protein distribution was determined using immunohistochemistry and Western blot. Their potential as biomarkers was finally assessed using enzyme-linked immunosorbent assay (ELISA) in cerebrospinal fluid (CSF) of scrapie sheep and Creutzfeldt-Jakob disease (CJD) patients. Protein BAMBI was upregulated in highly affected brain areas and CHGA was overexpressed along the brain in both models. Moreover, BAMBI and CHGA immunostaining scores strongly correlated with spongiosis and microgliosis in mice. Finally, levels of BAMBI were significantly higher in the CSF of clinical sheep and CJD patients. In addition to their potential as biomarkers, our work confirms the role of BAMBI and CHGA in prion neuropathology in vivo, but besides prion replication, they seem to be involved in the characteristic neuroinflammatory response associated to prion infection.

Poly (ADP-ribose) induces α-synuclein aggregation in neuronal-like cells and interacts with phosphorylated α-synuclein in post mortem PD samples

BackgroundPoly (ADP-ribose) (PAR) is a negatively charged polymer that is biosynthesized by Poly (ADP-ribose) Polymerase-1 (PARP-1) and regulates various cellular processes. Alpha-synuclein (αSyn) is an intrinsically disordered protein (IDP) that has been directly implicated with driving the onset and progression of Parkinson’s disease (PD). The mechanisms by which αSyn elicits its neurotoxic effects remain unclear. Recent findings indicate that one of the key processes driving PD pathology are oligomeric species of αSyn. Furthermore, it is well established that the main components of Lewy bodies (LBs) and Lewy neurites (LNs) in PD patients are aggregated hyperphosphorylated (S129) forms of αSyn (pαSyn).MethodsWe used biochemical and immunofluorescence-based assays to explore if PARP-1 enzymatic product (PAR) drives the conversion of monomeric αSyn into aggregated assemblies. We performed quantitative measurements using in situ proximity ligation assays (PLA) on a transgenic murine model of α-synucleinopathy (M83-SNCA*A53T) and post-mortem PD/PDD patient samples to characterize PAR-pαSyn interactions. Additionally, we used bioinformatic approaches and site-directed mutagenesis to identify PAR-binding regions on fibrillar αSyn.ResultsOur studies show that elevated intracellular levels of PAR promote the transition of αSyn into higher molecular weight forms. We report that PAR-pαSyn interactions are predominant in pathological states. Moreover, we confirm that the interactions between PAR and αSyn involve electrostatic forces between negatively charged PAR and lysine residues on the N-terminal region of αSyn.ConclusionsPAR plays a critical role in the early stages of monomeric αSyn aggregation, thereby attributing to PD pathogenesis. Based on our results, we report that PAR seeds monomeric αSyn aggregation and directly interacts with phosphorylated αSyn in conditions that are pathologically relevant to PD.

Chronic Oral Palmitoylethanolamide Administration Rescues Cognitive Deficit and Reduces Neuroinflammation, Oxidative Stress, and Glutamate Levels in A Transgenic Murine Model of Alzheimer’s Disease

N-palmitoylethanolamide (PEA) is a lipid mediator belonging to the class of the N-acylethanolamine. Products containing PEA, also in ultramicronized formulation (um-PEA), are already licensed for use in humans for its analgesic and anti-inflammatory properties, and demonstrated high safety and tolerability. Preclinical studies indicate that PEA, especially in the ultramicronized form, could be a potential therapeutic agent for Alzheimer’s disease (AD). In this study, we evaluated the neuroprotective and antioxidant effects of chronic (three months) um-PEA administration in an animal model of AD (3×Tg-AD mice). For translation purposes, the compound has been orally administered. Cognitive performance as well as biochemical markers [(interleukin-16 (IL-16) and tumor necrosis factor-α (TNF-α)] levels, reactive oxygen species (ROS) production, synaptophysin and glutamate levels) have been evaluated at the end of um-PEA treatment. The results indicate that orally administered um-PEA was adsorbed and distributed in the mice brain. The chronic treatment with um-PEA (100 mg/kg/day for three months) rescued cognitive deficit, restrained neuroinflammation and oxidative stress, and reduced the increase in hippocampal glutamate levels observed in 3×Tg-AD mice. Overall, these data reinforce the concept that um-PEA exerts beneficial effects in 3×Tg-AD mice. The fact that PEA is already licensed for the use in humans strongly supports its rapid translation in clinical practice.

Sensitization of nociceptors by prostaglandin E2–glycerol contributes to hyperalgesia in mice with sickle cell disease

Pain is a characteristic feature of sickle cell disease (SCD), 1 of the most common inherited diseases. Patients may experience acute painful crises as well as chronic pain. In the Berkley transgenic murine model of SCD, HbSS-BERK mice express only human hemoglobin S. These mice share many features of SCD patients, including persistent inflammation and hyperalgesia. Cyclooxygenase-2 (COX-2) is elevated in skin, dorsal root ganglia (DRG), and spinal cord in HbSS-BERK mice. In addition to arachidonic acid, COX-2 oxidizes the endocannabinoid 2-arachidonoylglycerol (2-AG) to produce prostaglandin E2 (PGE2)–glycerol (PGE2-G); PGE2-G is known to produce hyperalgesia. We tested the hypotheses that PGE2-G is increased in DRGs of HbSS-BERK mice and sensitizes nociceptors (sensory neurons that respond to noxious stimuli), and that blocking its synthesis would decrease hyperalgesia in HbSS-BERK mice. Systemic administration of R-flurbiprofen preferentially reduced production of PGE2-G over that of PGE2 in DRGs, decreased mechanical and thermal hyperalgesia, and decreased sensitization of nociceptors in HbSS-BERK mice. The same dose of R-flurbiprofen had no behavioral effect in HbAA-BERK mice (the transgenic control), but local injection of PGE2-G into the hind paw of HbAA-BERK mice produced sensitization of nociceptors and hyperalgesia. Coadministration of a P2Y6 receptor antagonist blocked the effect of PGE2-G, indicating that this receptor is a mediator of pain in SCD. The ability of R-flurbiprofen to block the synthesis of PGE2-G and to normalize levels of 2-AG suggests that R-flurbiprofen may be beneficial to treat pain in SCD, thereby reducing the use of opioids to relieve pain.

Simultaneous mesoscopic Ca2+ imaging and fMRI: Neuroimaging spanning spatiotemporal scales

ABSTRACTTo achieve a more comprehensive understanding of brain function requires simultaneous measurement of activity across a range of spatiotemporal scales. However, the appropriate tools to perform such studies are largely unavailable. Here, we present a novel approach for concurrent wide-field optical and functional magnetic resonance imaging (fMRI). By merging these two modalities, we are for the first time able to simultaneously acquire whole-brain blood-oxygen-level-dependent and whole-cortex calcium-sensitive fluorescent measures of brain activity. We describe the developments that allow us to combine these modalities without compromising the fidelity of either technique. In a transgenic murine model, we examine correspondences between activity measured using these modalities and identify unique and complementary features of each. Our approach links cell-type specific optical measurements of neural activity to the most widely used method for assessing human brain function. These data and approach directly establish the neural basis for the macroscopic connectivity patterns observed with fMRI.