Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

Regulation of GABA release by depolarisation-evoked Ca2+ transients at a single hippocampal terminal

We correlated dynamic changes in free cytosolic [Ca²⁺] ([Ca²⁺]_i) within single presynaptic terminals of cultured hippocampal neurones with the postsynaptic GABA-mediated currents. The local changes in [Ca²⁺]_i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp respectively. The Ca²⁺ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarising pulses. The presynaptic Ca²⁺ transient was graded by varying the amplitude of extracellular stimulating pulses. The probability of the release event, P, estimated for each stimulation strength, reached a maximum (P=1) when the Ca²⁺ signal became maximal and remained at this level at higher stimulation strength, despite the subsequent decrease in the amplitude of the Ca²⁺ transient. A gradual, linear increase in stimulation amplitude (V_stim) resulted in a bell-shaped dependence of the averaged amplitudes of Ca²⁺ signals and corresponding averaged amplitudes of eIPSCs. Analysis of the eIPSC demonstrated that the decrease in both the mean eIPSC amplitude and the mean quantal content of release resulted from a reduction in the probability of multivesicular release, i.e. in the disappearance of failures and in the decrease of individual eIPSC amplitude. The Ca²⁺ signals of similar amplitude resulted in both random and determinate (non-random) neurotransmitter release. We conclude that depolarisation-induced elevation of [Ca²⁺]_i within the terminal is necessary but not sufficient for activation of vesicular release of neurotransmitter.     

Activation of mouse microglial cells affects P2 receptor signaling

Microglial cells are the immunocompetent cells of the CNS, which are known to exist in several activation states. Here we investigated the impact of microglial activation on the P2 receptor-mediated intracellular calcium ([Ca(2+)](i)) signaling by means of fluo-3 based Ca(2+)-imaging. Cultured mouse microglial cells were treated with either astrocyte-conditioned medium to induce a ramified morphology or LPS to shift the cells toward the fully activated stage. The extracellular application of ATP (100 microM) induced a [Ca(2+)](i) elevation in 85% of both untreated and ramified microglial cells, whereas only 50% of the LPS-activated cells responded to the stimulus. To characterise the pharmacological profile of microglial P2 receptors we investigated the effects of various P2 agonists on [Ca(2+)](i) in cultured microglial cells. Untreated and ramified microglial cells demonstrated a very similar sensitivity to the different P2 agonists. In contrast, in LPS-activated microglia, a sharp decrease of responses to P2 agonist stimulation was seen. This indicates that microglial activation influences the capability of microglial cells to generate [Ca(2+)](i) signals upon P2 receptor activation.     

Neuronal ageing in long-term cultures: alterations of Ca2+ homeostasis

Deficiencies of Ca2+ homeostasis are proposed to play an important role in neuronal ageing and/or neurodegeneration. The aim of this study was to investigate, in a defined neuronal population, primary cerebellar granule neuron culture, the time-dependent changes in Ca2+ homeostasis and compare them with data obtained in cerebellar brain slices from aged rats. In neurons aged in culture (DIV 23), a small decrease in the resting [Ca2+]i was associated with a decrease in the maximal rate of [Ca2+]i increase upon KCl-induced depolarization and in the amplitude of the [Ca2+]i response, when compared with mature neurons (DIV 9). The most significant change of [Ca2+]i signal parameters was a 50% decrease in the rate of [Ca2+]i recovery after the stimulation. These results were similar to those obtained in aged brain slices, and indicate that primary neuronal cultures could serve as a model for studying the age-related changes in Ca2+ homeostasis.     

Gene expression changes in dorsal root ganglia following peripheral nerve injury: roles in inflammation,cell death and nociception

Subsequent to a peripheral nerve injury, there are changes in gene expression within the dorsal root ganglia in response to the damage. This review selects factors which are well-known to be vital for inflammation, cell death and nociception, and highlights how alterations in their gene expression within the dorsal root ganglia can affect functional recovery. The majority of studies used polymerase chain reaction within animal models to analyse the dynamic changes following peripheral nerve injuries. This review aims to highlight the factors at the gene expression level that impede functional recovery and are hence are potential targets for therapeutic approaches. Where possible the experimental model, specific time-points and cellular location of expression levels are reported.     

Pathological Role for Exocytotic Glutamate Release from Astrocytes in Hepatic Encephalopathy

Liver failure can lead to generalized hyperammonemia, which is thought to be the underlying cause of hepatic encephalopathy. This neuropsychiatric syndrome is accompanied by functional changes of astrocytes. These glial cells enter ammonia-induced self-amplifying cycle characterized by brain oedema, oxidative and osmotic stress that causes modification of proteins and RNA. Consequently, protein expression and function are affected, including that of glutamine synthetase and plasmalemmal glutamate transporters, leading to glutamate excitotoxicity; Ca²⁺-dependent exocytotic glutamate release from astrocytes contributes to this extracellular glutamate overload.     

Channelrhodopsin-2–XXL,a powerful optogenetic tool for low-light applications

Channelrhodopsin-2 (ChR2) has provided a breakthrough for the optogenetic control of neuronal activity. In adult Drosophila melanogaster, however, its applications are severely constrained. This limitation in a powerful model system has curtailed unfolding the full potential of ChR2 for behavioral neuroscience. Here, we describe the D156C mutant, termed ChR2-XXL (extra high expression and long open state), which displays increased expression, improved subcellular localization, elevated retinal affinity, an extended open-state lifetime, and photocurrent amplitudes greatly exceeding those of all heretofore published ChR variants. As a result, neuronal activity could be efficiently evoked with ambient light and even without retinal supplementation. We validated the benefits of the variant in intact flies by eliciting simple and complex behaviors. We demonstrate efficient and prolonged photostimulation of monosynaptic transmission at the neuromuscular junction and reliable activation of a gustatory reflex pathway. Innate male courtship was triggered in male and female flies, and olfactory memories were written through light-induced associative training.Identifying causal relationships between neuronal activity and animal behavior is a fundamental goal of neuroscience. Crucially, this task requires testing whether defined neuronal populations are sufficient for eliciting behavioral modules. The development of light-gated ion channels that can be genetically targeted to specific cells has provided a unique solution to this challenge. In pioneering work, such optogenetic effectors or actuators were originally used as multicomponent approaches (1–3). The introduction of Channelrhodopsin-1 (ChR1) (4) and especially ChR2 as a light-sensitive cation channel (5) dramatically advanced the field by providing an efficient and straightforward single-component strategy for stimulating neuronal activity (6, 7).Besides cell-specific targeting of appropriate effector elements, precise neuronal control by optogenetics demands efficient light delivery to the neurons of interest. For behavioral studies, photostimulation is ideally accomplished in intact, freely moving organisms and accompanied by functional readouts. The combination of a rich, well-characterized behavioral repertoire and elegant molecular genetics has contributed to Drosophila’s strong impact on behavioral neurogenetics (8, 9). However, low light transmission through the pigmented cuticle presupposes high light intensities for using ChR2 in flies. This obstacle greatly complicates the experimental setup for freely moving animals, and the required light energies can cause heat damage when stimulation is applied over extended time periods. Moreover, limited cellular availability of all-trans-retinal (hereafter retinal for short) demands adding high retinal concentrations as a dietary supplement. If optical access to target cells is not provided by a translucent body wall (e.g., as in nematodes, zebrafish, and Drosophila larvae), an alternative solution is the implantation of an optical fiber directly into the brain. Although this approach has been used successfully in mammals (10), such an invasive procedure is infeasible for the study of intact small organisms.Due to these restrictions in Drosophila, ChR2 has not reached the popularity attained in other organisms, and instead the field has turned mainly to thermogenetic neuronal stimulation (11–13). As with all techniques, there are also drawbacks to using temperature as a stimulus, such as undesired background activity and a multitude of temperature-sensitive cellular processes and behavioral responses. Photo-liberation of caged ATP, combined with genetic targeting of ATP-gated ion channels, has been introduced as a different optogenetic technique in Drosophila (3, 14). However, its applications are constrained by invasive, time-consuming procedures for injection of caged ATP and a limited experimental time window.Here, we introduce improved ChR2 variants as an alternative approach to address these shortcomings in Drosophila. Compared with wild-type ChR2 (ChR2-wt), expression of these mutants in target cells led to strongly enhanced photocurrents. We provide the first report, to our knowledge, of ChR2-T159C (15, 16) in flies and describe a ChR2 variant, ChR2-XXL (extra high expression and long open state), that is characterized by an extended open-state lifetime, elevated cellular expression, enhanced axonal localization, and reduced dependence on retinal addition. As a consequence, this mutant does not require dietary retinal supplementation to depolarize cells, evoke synaptic transmission, and activate neuronal networks at very low irradiance. These features enabled behavioral photostimulation in freely moving flies using diffuse low-intensity light.     

Regulatory element copy number differences shape primate expression profiles

Gene expression differences are shaped by selective pressures and contribute to phenotypic differences between species. We identified 964 copy number differences (CNDs) of conserved sequences across three primate species and examined their potential effects on gene expression profiles. Samples with copy number different genes had significantly different expression than samples with neutral copy number. Genes encoding regulatory molecules differed in copy number and were associated with significant expression differences. Additionally, we identified 127 CNDs that were processed pseudogenes and some of which were expressed. Furthermore, there were copy number-different regulatory regions such as ultraconserved elements and long intergenic noncoding RNAs with the potential to affect expression. We postulate that CNDs of these conserved sequences fine-tune developmental pathways by altering the levels of RNA.