Neuronal nuclear calcium signaling suppression of microglial reactivity is mediated by osteoprotegerin after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is characterized by massive changes in neuronal excitation, from acute excitotoxicity to chronic hyper- or hypoexcitability. Nuclear calcium signaling pathways are involved in translating changes in synaptic inputs and neuronal activity into discrete transcriptional programs which not only affect neuronal survival and synaptic integrity, but also the crosstalk between neurons and glial cells. Here, we report the effects of blunting neuronal nuclear calcium signals in the context of TBI. METHODS: We used AAV vectors to express the genetically encoded and nuclear-targeted calcium buffer parvalbumin (PV.NLS.mCherry) or the calcium/calmodulin buffer CaMBP4.mCherry in neurons only. Upon TBI, the extent of neuroinflammation, neuronal death and synaptic loss were assessed by immunohistochemistry and targeted transcriptome analysis. Modulation of the overall level of neuronal activity was achieved by PSAM/PSEM chemogenetics targeted to parvalbumin interneurons. The functional impact of neuronal nuclear calcium buffering in TBI was assessed by quantification of spontaneous whisking. RESULTS: Buffering neuronal nuclear calcium unexpectedly resulted in a massive and long-lasting increase in the recruitment of reactive microglia to the injury site, which was characterized by a disease-associated and phagocytic phenotype. This effect was accompanied by a substantial surge in synaptic loss and significantly reduced whisking activity. Transcriptome analysis revealed a complex effect of TBI in the context of neuronal nuclear calcium buffering, with upregulation of complement factors, chemokines and interferon-response genes, as well as the downregulation of synaptic genes and epigenetic regulators compared to control conditions. Notably, nuclear calcium buffering led to a substantial loss in neuronal osteoprotegerin (OPG), whereas stimulation of neuronal firing induced OPG expression. Viral re-expression of OPG resulted in decreased microglial recruitment and synaptic loss. OPG upregulation was also observed in the CSF of human TBI patients, underscoring its translational value. CONCLUSION: Neuronal nuclear calcium signals regulate the degree of microglial recruitment and reactivity upon TBI via, among others, osteoprotegerin signals. Our findings support a model whereby neuronal activity altered after TBI exerts a powerful impact on the neuroinflammatory cascade, which in turn contributes to the overall loss of synapses and functional impairment.

Mapping the adult human esophagus in vivo and in vitro.

Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus.

Shank2/3 double knockout-based screening of cortical subregions links the retrosplenial area to the loss of social memory in autism spectrum disorders.

Members of the Shank protein family are master scaffolds of the postsynaptic architecture and mutations within the SHANK genes are causally associated with autism spectrum disorders (ASDs). We generated a Shank2-Shank3 double knockout mouse that is showing severe autism related core symptoms, as well as a broad spectrum of comorbidities. We exploited this animal model to identify cortical brain areas linked to specific autistic traits by locally deleting Shank2 and Shank3 simultaneously. Our screening of 10 cortical subregions revealed that a Shank2/3 deletion within the retrosplenial area severely impairs social memory, a core symptom of ASD. Notably, DREADD-mediated neuronal activation could rescue the social impairment triggered by Shank2/3 depletion. Data indicate that the retrosplenial area has to be added to the list of defined brain regions that contribute to the spectrum of behavioural alterations seen in ASDs.

Disruption of orbitofrontal-hypothalamic projections in a murine ALS model and in human patients.

BACKGROUND: Increased catabolism has recently been recognized as a clinical manifestation of amyotrophic lateral sclerosis (ALS). The hypothalamic systems have been shown to be involved in the metabolic dysfunction in ALS, but the exact extent of hypothalamic circuit alterations in ALS is yet to be determined. Here we explored the integrity of large-scale cortico-hypothalamic circuits involved in energy homeostasis in murine models and in ALS patients. METHODS: The rAAV2-based large-scale projection mapping and image analysis pipeline based on Wholebrain and Ilastik software suites were used to identify and quantify projections from the forebrain to the lateral hypothalamus in the SOD1(G93A) ALS mouse model (hypermetabolic) and the FusΔNLS ALS mouse model (normo-metabolic). 3 T diffusion tensor imaging (DTI)-magnetic resonance imaging (MRI) was performed on 83 ALS and 65 control cases to investigate cortical projections to the lateral hypothalamus (LHA) in ALS. RESULTS: Symptomatic SOD1(G93A) mice displayed an expansion of projections from agranular insula, ventrolateral orbitofrontal and secondary motor cortex to the LHA. These findings were reproduced in an independent cohort by using a different analytic approach. In contrast, in the FusΔNLS ALS mouse model hypothalamic inputs from insula and orbitofrontal cortex were maintained while the projections from motor cortex were lost. The DTI-MRI data confirmed the disruption of the orbitofrontal-hypothalamic tract in ALS patients. CONCLUSION: This study provides converging murine and human data demonstrating the selective structural disruption of hypothalamic inputs in ALS as a promising factor contributing to the origin of the hypermetabolic phenotype.

Multiplexed chemogenetics in astrocytes and motoneurons restore blood-spinal cord barrier in ALS.

Blood-spinal cord barrier (BSCB) disruption is thought to contribute to motoneuron (MN) loss in amyotrophic lateral sclerosis (ALS). It is currently unclear whether impairment of the BSCB is the cause or consequence of MN dysfunction and whether its restoration may be directly beneficial. We revealed that SOD1 G93A , FUS ΔNLS , TDP43 G298S , and Tbk1 +/- ALS mouse models commonly shared alterations in the BSCB, unrelated to motoneuron loss. We exploit PSAM/PSEM chemogenetics in SOD1 G93A mice to demonstrate that the BSCB is rescued by increased MN firing, whereas inactivation worsens it. Moreover, we use DREADD chemogenetics, alone or in multiplexed form, to show that activation of Gi signaling in astrocytes restores BSCB integrity, independently of MN firing, with no effect on MN disease markers and dissociating them from BSCB disruption. We show that astrocytic levels of the BSCB stabilizers Wnt7a and Wnt5a are decreased in SOD1 G93A mice and strongly enhanced by Gi signaling, although further decreased by MN inactivation. Thus, we demonstrate that BSCB impairment follows MN dysfunction in ALS pathogenesis but can be reversed by Gi-induced expression of astrocytic Wnt5a/7a.

Synaptic restoration by cAMP/PKA drives activity-dependent neuroprotection to motoneurons in ALS.

Excessive excitation is hypothesized to cause motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS), but actual proof of hyperexcitation in vivo is missing, and trials based on this concept have failed. We demonstrate, by in vivo single-MN electrophysiology, that, contrary to expectations, excitatory responses evoked by sensory and brainstem inputs are reduced in MNs of presymptomatic mutSOD1 mice. This impairment correlates with disrupted postsynaptic clustering of Homer1b, Shank, and AMPAR subunits. Synaptic restoration can be achieved by activation of the cAMP/PKA pathway, by either intracellular injection of cAMP or DREADD-Gs stimulation. Furthermore, we reveal, through independent control of signaling and excitability allowed by multiplexed DREADD/PSAM chemogenetics, that PKA-induced restoration of synapses triggers an excitation-dependent decrease in misfolded SOD1 burden and autophagy overload. In turn, increased MN excitability contributes to restoring synaptic structures. Thus, the decrease of excitation to MN is an early but reversible event in ALS. Failure of the postsynaptic site, rather than hyperexcitation, drives disease pathobiochemistry.

Stage-dependent remodeling of projections to motor cortex in ALS mouse model revealed by a new variant retrograde-AAV9.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of motoneurons in the primary motor cortex (pMO) and in spinal cord. However, the pathogenic process involves multiple subnetworks in the brain and functional MRI studies demonstrate an increase in functional connectivity in areas connected to pMO despite the ongoing neurodegeneration. The extent and the structural basis of the motor subnetwork remodeling in experimentally tractable models remain unclear. We have developed a new retrograde AAV9 to quantitatively map the projections to pMO in the SOD1(G93A) ALS mouse model. We show an increase in the number of neurons projecting from somatosensory cortex to pMO at presymptomatic stages, followed by an increase in projections from thalamus, auditory cortex and contralateral MO (inputs from 20 other structures remains unchanged) as disease advances. The stage- and structure-dependent remodeling of projection to pMO in ALS may provide insights into the hyperconnectivity observed in ALS patients.