Phospholipid-flippase chaperone CDC50A is required for synapse maintenance by regulating phosphatidylserine exposure.

Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.

GPR56 S4 variant is required for microglia-mediated synaptic pruning.

ADGRG1 (also called GPR56) plays critical roles in brain development and wiring, including cortical lamination, central nervous system (CNS) myelination, and developmental synaptic refinement. However, the underlying mechanism(s) in mediating such diverse functions is not fully understood. Here, we investigate the function of one specific alternative splicing isoform, the GPR56 splice variant 4 (S4), to test the hypothesis that alternative splicing variants of GPR56 in part support its different functions. We created a new transgenic mouse line, Gpr56∆S4 , using CRISPR/Cas9, in which GPR56 S4 was deleted. Detailed phenotype analyses show that Gpr56∆S4 mice manifest no deficits in cortical architecture and CNS myelination compared to controls. Excitingly, they present significantly increased synapse densities, decreased synapse engulfment by microglia, and impaired eye-segregation. Taken together, our findings support that the GPR56 S4 variant is dispensable for cortical development and CNS myelination but is essential for microglia-mediated synaptic pruning.

GAIN domain-mediated cleavage is required for activation of G protein-coupled receptor 56 (GPR56) by its natural ligands and a small-molecule agonist.

Adhesion G protein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members.

Marine bacterial extracts as a new rich source of drugs against Alzheimer's disease.

Alzheimer's disease (AD) is a prevalent, progressive and irreversible, neurodegenerative disease with no disease modifying treatment yet available. The projected burden of AD on our healthcare system is immense and thus there is an immediate need for new drugs that prevent or attenuate AD symptoms. While most efforts in the field are directed at treatments that reduce amyloid or tau burden in the brain, we have taken an alternate approach - a model based on reducing AD-associated neuronal cell cycle events. Using this model, we have screened a largely unexplored source of compounds with therapeutic potential - the natural products created by diverse strains of marine bacteria. Two hundred and twenty-five bacterial extracts from different strains were tested for both toxicity and neuroprotective properties by crystal violet and In-cell Western - first in HT22 cells and then in mouse primary neuronal cultures. Based on these screens, we have identified several promising leads, and here we focus on the most promising of these. We found that we could directly assay even a crude bacterial extract in our E16 mouse cortical neuronal cultures and screen for activities that prevent cell cycle reentry and preserve synaptic structure. Preliminary tests in 1-month-old animals from a mouse model of Ataxia telangiectasia, showed that blockage of cell cycle-related neuronal death could also be successful in vivo. This adds an important extension to our in vitro studies. These findings showcase a new effective and efficient assay system and validate the use of marine natural compounds as a novel source for new drugs to fight Alzheimer's disease. Cover Image for this issue: doi: 10.1111/jnc.14733.

Testing the Neuroprotective Properties of PCSO-524® Using a Neuronal Cell Cycle Suppression Assay.

Cell cycle reentry is a unified mechanism shared by several neurodegenerative diseases, including Alzheimer's disease (AD) and Ataxia Telangiectasia (A-T). This phenotype is often related to neuroinflammation in the central nervous system. To mimic brain inflammation in vitro, we adopted the previously established method of using conditioned medium collected from activated THP-1 cells and applied it to both differentiated HT22 cells and primary neurons. Unscheduled cell cycle events were observed in both systems, indicating the potential of this approach as an in vitro model of neurodegenerative disease. We used this assay to measure the neuroprotective effects of New Zealand green-lipped mussel extract, PCSO-524®, to protect post-mitotic cells from cell cycle reentry. We found that, both in vitro and in an animal model, PCSO-524® displayed promising neuroprotective effects, and thus has potential to postpone or prevent the onset of neurodegenerative disease.

Family-wide Structural Characterization and Genomic Comparisons Decode the Diversity-oriented Biosynthesis of Thalassospiramides by Marine Proteobacteria.

The thalassospiramide lipopeptides have great potential for therapeutic applications; however, their structural and functional diversity and biosynthesis are poorly understood. Here, by cultivating 130 Rhodospirillaceae strains sampled from oceans worldwide, we discovered 21 new thalassospiramide analogues and demonstrated their neuroprotective effects. To investigate the diversity of biosynthetic gene cluster (BGC) architectures, we sequenced the draft genomes of 28 Rhodospirillaceae strains. Our family-wide genomic analysis revealed three types of dysfunctional BGCs and four functional BGCs whose architectures correspond to four production patterns. This correlation allowed us to reassess the "diversity-oriented biosynthesis" proposed for the microbial production of thalassospiramides, which involves iteration of several key modules. Preliminary evolutionary investigation suggested that the functional BGCs could have arisen through module/domain loss, whereas the dysfunctional BGCs arose through horizontal gene transfer. Further comparative genomics indicated that thalassospiramide production is likely to be attendant on particular genes/pathways for amino acid metabolism, signaling transduction, and compound efflux. Our findings provide a systematic understanding of thalassospiramide production and new insights into the underlying mechanism.

Microglial GPR56 is the molecular target of maternal immune activation-induced parvalbumin-positive interneuron deficits.

Parvalbumin-positive (PV+) interneurons play a critical role in maintaining circuit rhythm in the brain, and their reduction is implicated in autism spectrum disorders. Animal studies demonstrate that maternal immune activation (MIA) leads to reduced PV+ interneurons in the somatosensory cortex and autism-like behaviors. However, the underlying molecular mechanisms remain largely unknown. Here, we show that MIA down-regulates microglial Gpr56 expression in fetal brains in an interleukin-17a-dependent manner and that conditional deletion of microglial Gpr56 [Gpr56 conditional knockout (cKO)] mimics MIA-induced PV+ interneuron defects and autism-like behaviors in offspring. We further demonstrate that elevated microglial tumor necrosis factor-α expression is the underlying mechanism by which MIA and Gpr56 cKO impair interneuron generation. Genetically restoring Gpr56 expression in microglia ameliorates PV+ interneuron deficits and autism-like behaviors in MIA offspring. Together, our study demonstrates that microglial GPR56 plays an important role in PV+ interneuron development and serves as a salient target of MIA-induced neurodevelopmental disorders.

Identifying a Population of Glial Progenitors That Have Been Mistaken for Neurons in Embryonic Mouse Cortical Culture.

Experiments in primary culture have helped advance our understanding of the curious phenomenon of cell cycle-related neuronal death. In a differentiated postmitotic cell such as a neuron, aberrant cell cycle reentry is strongly associated with apoptosis. Indeed, in many pathologic conditions, neuronal populations at risk for death are marked by cells engaged in a cell cycle like process. The evidence for this conclusion is typically based on finding MAP2+ cells that are also positive for cell cycle-related proteins (e.g., cyclin D) or have incorporated thymidine analogs such as bromodeoxyuridine (BrdU) or 5-ethynyl-2'-deoxyuridine (EdU) into their nuclei. We now report that we and others may have partly been led astray in pursuing this line of work. Morphometric analysis of mouse embryonic cortical cultures reveals that the size of the "cycling" MAP2+ cells is significantly smaller than those of normal neurons, and their expression of MAP2 is significantly lower. This led us to ask whether, rather than representing fully developed neurons, they more closely resembled precursor-like cells. In support of this idea, we find that these small MAP2+ cells are immunopositive for nestin, a neuronal precursor marker, Olig2, an oligodendrocyte lineage marker, and neural/glial antigen 2 (NG2), an oligodendrocyte precursor marker. Tracking their behavior in culture, we find that they predominantly give rise to GFAP+ astrocytes instead of neurons or oligodendrocytes. These findings argue for a critical reexamination of previous reports of stimuli that lead to neuronal cell cycle-related death in primary cultures.