Neuronal circuit assembly requires the fine balance between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM 2 and the soluble complement proteins C1q and C3 are recognized as key players, the neuronal molecular components that specify synapses to be eliminated are still undefined. Here, we show that exposed phosphatidylserine ( PS ) represents a neuronal “eat‐me” signal involved in microglial‐mediated pruning. In hippocampal neuron and microglia co‐cultures, synapse elimination can be partially prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM 2. In vivo , PS exposure at both hippocampal and retinogeniculate synapses and engulfment of PS ‐labeled material by microglia occurs during established developmental periods of microglial‐mediated synapse elimination. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, have elevated presynaptic PS exposure and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial‐mediated synapse pruning and identify a novel role of developmentally regulated neuronal PS exposure that is common among developing brain structures.

Neuronal circuits assembly requires the fine equilibrium between synapse formation and elimination. Microglia, through the elimination of supernumerary synapses, have an established role in this process. While the microglial receptor TREM2 and the soluble complement proteins C1q and C3 are recognized key players in this process, the neuronal molecular components that tag synapses to be eliminated are still undefined. Here we show that exposed phosphatidylserine (PS) represents a neuronal 'eat-me' signal enabling microglial-mediated synapse pruning. In hippocampal neuron and microglia co-cultures, synapse elimination can be prevented by blocking accessibility of exposed PS using Annexin V or through microglial loss of TREM2. In vivo, exposed PS is detectable at both hippocampal and retinogeniculate synapses, where exposure coincides with the onset of synapse elimination and increased PS engulfment by microglia. Mice deficient in C1q, which fail to properly refine retinogeniculate connections, display elevated exposed PS and reduced PS engulfment by microglia. These data provide mechanistic insight into microglial-mediated synapse pruning and identify a novel role of developmentally regulated PS exposure that is common among developing brain structures.

Mutations in the CDKL5 gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 pathogenic mutations are candidates to affect splicing, we evaluated the capability of variants of the spliceosomal U1 small nuclear RNA (U1snRNA) to correct mutations affecting +1 and +5 nucleotides at the 5′ donor splice site and predicted to cause exon skipping. Our results show that CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 splicing pattern. The expression of engineered U1snRNA effectively rescued mutations at +5 but not at the +1 nucleotides. Importantly, we proved that U1snRNA-mediated splicing correction fully restores CDKL5 protein synthesis, subcellular distribution and kinase activity. Eventually, by correcting aberrant splicing of an exogenously expressed splicing-competent CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.

The role of microglia in controlling synapse homeostasis is becoming increasingly recognized by the scientific community. In particular, the microglia-mediated elimination of supernumerary synapses during development lays the basis for the correct formation of neuronal circuits in adulthood, while the possible reactivation of this process in pathological conditions, such as schizophrenia or Alzheimer's Disease, provides a promising target for future therapeutic strategies. The methodological approaches to investigate microglial synaptic engulfment include different in vitro and in vivo settings. Basic in vitro assays, employing isolated microglia and microbeads, apoptotic membranes, liposomes or synaptosomes allow the quantification of the microglia phagocytic abilities, while co-cultures of microglia and neurons, deriving from either WT or genetically modified mice models, provide a relatively manageable setting to investigate the involvement of specific molecular pathways. Further detailed analysis in mice brain is then mandatory to validate the in vitro assays as representative for the in vivo situation. The present review aims to dissect the main technical approaches to investigate microglia-mediated phagocytosis of neuronal and synaptic substrates in critical developmental time windows.

Background and ObjectivesCDKL5 deficiency disorder (CDD) is a neurodevelopmental encephalopathy characterized by early-onset epilepsy and impaired psychomotor development. Variations in the X-linked CDKL5 gene coding for a kinase cause CDD. Molecular genetics has proved that almost all pathogenic missense substitutions localize in the N-terminal catalytic domain, therefore underlining the importance for brain development and functioning of the kinase activity. CDKL5 also features a long C-terminal domain that acts as negative regulator of the enzymatic activity and modulates its subcellular distribution. CDD is generally attributed to loss-of-function variations, whereas the clinical consequences of increased CDKL5 activity remain uncertain. We have identified a female patient characterized by mild epilepsy and neurologic symptoms, harboring a novel c.2873C>G nucleotide substitution, leading to the missense variant p.(Thr958Arg). To increase our comprehension of genetic variants in CDKL5-associated neurologic disorders, we have characterized the molecular consequences of the identified substitution.MethodsMRI and video EEG telemetry were used to describe brain activity and capture seizure. The Bayley III test was used to evaluate the patient development. Reverse transcriptase PCR was used to analyze whether the identified nucleotide variant affects messenger RNA stability and/or splicing. The X chromosome inactivation pattern was analyzed determining the DNA methylation status of the androgen receptor (AR) gene and by sequencing of expressed alleles. Western blotting was used to investigate whether the novel Thr958Arg substitution affects the stability and/or enzymatic activity of CDKL5. Immunofluorescence was used to define whether CDKL5 subcellular distribution is affected by the Thr958Arg substitution.ResultsOur data suggested that the proband tends toward a skewed X chromosome inactivation pattern in favor of the novel variant. The molecular investigation revealed that the p.(Thr958Arg) substitution leads to a significant increase in the autophosphorylation of both the TEY motif and residue Tyr171 of CDKL5, as well as in the phosphorylation of the target protein MAP1S, indicating an hyperactivation of CDKL5. This occurs without evidently affecting the kinase subcellular distribution.DiscussionOur data provide a strong indication that the c.2873C>G nucleotide substitution represents an hypermorphic pathogenic variation of CDKL5, therefore highlighting the importance of a tight control of CDKL5 activity in the brain.