MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation.

Correlative evidence has suggested that the methyl-CpG-binding protein MeCP2 contributes to the formation of heterochromatin condensates via liquid-liquid phase separation. This interpretation has been reinforced by the observation that heterochromatin, DNA methylation and MeCP2 co-localise within prominent foci in mouse cells. The findings presented here revise this view. MeCP2 localisation is independent of heterochromatin as MeCP2 foci persist even when heterochromatin organisation is disrupted. Additionally, MeCP2 foci fail to show hallmarks of phase separation in live cells. Importantly, we find that mouse cellular models are highly atypical as MeCP2 distribution is diffuse in most mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which is a mouse subspecies lacking methylated satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form condensates and its localisation is independent of heterochromatin. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns.

Comparative analysis of potential broad-spectrum neuronal Cre drivers.

Cre/Lox technology is a powerful tool in the mouse genetics tool-box as it enables tissue-specific and inducible mutagenesis of specific gene loci. Correct interpretation of phenotypes depends upon knowledge of the Cre expression pattern in the chosen mouse driver line to ensure that appropriate cell types are targeted. For studies of the brain and neurological disease a pan-neuronal promoter that reliably drives efficient neuron-specific transgene expression would be valuable. Here we compare a widely used "pan-neuronal" mouse Cre driver line, Syn1-cre, with a little-known alternative, Snap25-IRES2-cre. Our results show that the Syn1-cre line broadly expresses in the brain but is indetectable in more than half of all neurons and weakly active in testes. In contrast the Snap25-IRES2-cre line expressed Cre in a high proportion of neurons (~85%) and was indetectable in all non-brain tissues that were analysed, including testes. Our findings suggest that for many purposes Snap25-IRES2-cre is superior to Syn1-cre as a potential pan-neuronal cre driver.

To what extent are GCS and AVPU equivalent to each other when assessing the level of consciousness of children with head injury? A cross-sectional study of UK hospital admissions.

OBJECTIVE: To evaluate utility and equivalence of Glasgow Coma Scale (GCS) and the Alert, Voice, Pain, Unresponsive (AVPU) scale in children with head injury. DESIGN: Cross sectional study. SETTING: UK hospital admissions: September 2009-February 2010. PATIENTS: <15 years with head injury. INTERVENTIONS: GCS and/or AVPU at injury scene and in emergency departments (ED). MAIN OUTCOME: Measures used, the equivalence of AVPU to GCS, GCS at the scene predicting GCS in ED, CT results by age, hospital type. RESULTS: Level of consciousness was recorded in 91% (5168/5700) in ED (43%: GCS/30.5%: GCS+AVPU/17.3%: AVPU) and 66.1% (1190/1801) prehospital (33%: GCS/26%GCS+AVPU/7%: AVPU). Failure to record level of consciousness and the use of AVPU were greatest for infants. Correlation between AVPU and median GCS in 1147 children <5 years: A=15, V=14, P=8, U=3, for 1163 children ≥5 years: A=15, V=13, P=11, U=3. There was no significant difference in the proportion of infants who had a CT whether AVPU=V/P/U or GCS<15. However diagnostic yield of intracranial injury or depressed fracture was significantly greater for V/P/U than GCS<15 :7/7: 100% (95% CI 64.6% to 100%) versus 5/17: 29.4% (95% CI 13.3% to 53.1%). For children >1 year significantly more had a CT scan when GCS<14 was recorded than 'V/P/U only' and the diagnostic yield was greater. Prehospital GCS and GCS in the ED were the same for 77.4% (705/911). CONCLUSION: There was a clear correlation between Alert and GCS=15 and between Unresponsive and GCS=3 but a wider range of GCS scores for responsive to Pain or Voice that varied with age. AVPU was valuable at initial assessment of infants and did not adversely affect the proportion of infants who had head CT or the diagnostic yield.

Efficient and versatile CRISPR engineering of human neurons in culture to model neurological disorders.

The recent identification of multiple new genetic causes of neurological disorders highlights the need for model systems that give experimental access to the underlying biology. In particular, the ability to couple disease-causing mutations with human neuronal differentiation systems would be beneficial. Gene targeting is a well-known approach for dissecting gene function, but low rates of homologous recombination in somatic cells (including neuronal cells) have traditionally impeded the development of robust cellular models of neurological disorders. Recently, however, CRISPR/Cas9 gene editing technologies have expanded the number of systems within which gene targeting is possible. Here we adopt as a model system LUHMES cells, a commercially available diploid human female mesencephalic cell line that differentiates into homogeneous mature neurons in 1-2 weeks. We describe optimised methods for transfection and selection of neuronal progenitor cells carrying targeted genomic alterations using CRISPR/Cas9 technology. By targeting the endogenous X-linked MECP2 locus, we introduced four independent missense mutations that cause the autism spectrum disorder Rett syndrome and observed the desired genetic structure in 3-26% of selected clones, including gene targeting of the inactive X chromosome. Similar efficiencies were achieved by introducing neurodevelopmental disorder-causing mutations at the autosomal EEF1A2 locus on chromosome 20. Our results indicate that efficiency of genetic "knock-in" is determined by the location of the mutation within the donor DNA molecule. Furthermore, we successfully introduced an mCherry tag at the MECP2 locus to yield a fusion protein, demonstrating that larger insertions are also straightforward in this system. We suggest that our optimised methods for altering the genome of LUHMES cells make them an attractive model for the study of neurogenetic disorders.