SUMMARY
Massively parallel single-cell RNA sequencing can precisely resolve
cellular diversity in a high-throughput manner at low cost, but unbiased
isolation of intact single cells from complex tissues, such as adult mammalian
brains, is challenging. Here, we integrate sucrose-gradient assisted
purification of nuclei with droplet microfluidics to develop a highly scalable
single-nucleus RNA-Seq approach (sNucDrop-Seq), which is free of enzymatic
dissociation and nuclei sorting. By profiling ~18,000 nuclei isolated
from cortical tissues of adult mice, we demonstrate that sNucDrop-Seq not only
accurately reveals neuronal and non-neuronal subtype composition with high
sensitivity, but also enables in-depth analysis of transient transcriptional
states driven by neuronal activity, at single-cell resolution, in
vivo.
Cyclin-dependent kinase-like 5 (CDKL5) deficiency is a neurodevelopmental disorder characterized by epileptic seizures, severe intellectual disability, and autistic features. Mice lacking CDKL5 display multiple behavioral abnormalities reminiscent of the disorder, but the cellular origins of these phenotypes remain unclear. Here, we find that ablating CDKL5 expression specifically from forebrain glutamatergic neurons impairs hippocampal-dependent memory in male conditional knock-out mice. Hippocampal pyramidal neurons lacking CDKL5 show decreased dendritic complexity but a trend toward increased spine density. This morphological change is accompanied by an increase in the frequency of spontaneous miniature EPSCs and interestingly, miniature IPSCs. Using voltage-sensitive dye imaging to interrogate the evoked response of the CA1 microcircuit, we find that CA1 pyramidal neurons lacking CDKL5 show hyperexcitability in their dendritic domain that is constrained by elevated inhibition in a spatially and temporally distinct manner. These results suggest a novel role for CDKL5 in the regulation of synaptic function and uncover an intriguing microcircuit mechanism underlying impaired learning and memory.
CDKL5 deficiency disorder (CDD) is characterized by epilepsy, intellectual disability, and autistic features, and CDKL5-deficient mice exhibit a constellation of behavioral phenotypes reminiscent of the human disorder. We previously found that CDKL5 dysfunction in forebrain glutamatergic neurons results in deficits in learning and memory. However, the pathogenic origin of the autistic features of CDD remains unknown. Here, we find that selective loss of CDKL5 in GABAergic neurons leads to autistic-like phenotypes in mice accompanied by excessive glutamatergic transmission, hyperexcitability, and increased levels of postsynaptic NMDA receptors. Acute, low-dose inhibition of NMDAR signaling ameliorates autistic-like behaviors in GABAergic knockout mice, as well as a novel mouse model bearing a CDD-associated nonsense mutation, CDKL5 R59X, implicating the translational potential of this mechanism. Together, our findings suggest that enhanced NMDAR signaling and circuit hyperexcitability underlie autistic-like features in mouse models of CDD and provide a new therapeutic avenue to treat CDD-related symptoms.
Insulin-producing cells (IPCs) in the Drosophila brain produce and release insulin-like peptides (ILPs) to the hemolymph. ILPs are crucial for growth and regulation of metabolic activity in flies, functions analogous to those of mammalian insulin and insulin-like growth factors (IGFs). To identify components functioning in IPCs to control ILP production, we employed genomic and candidate gene approaches. We used laser microdissection and messenger RNA sequencing to characterize the transcriptome of larval IPCs. IPCs highly express many genes homologous to genes active in insulin-producing β-cells of the mammalian pancreas. The genes in common encode ILPs and proteins that control insulin metabolism, storage, secretion, β-cell proliferation, and some not previously linked to insulin production or β-cell function. Among these novelties is unc-104, a kinesin 3 family gene, which is more highly expressed in IPCs compared to most other neurons. Knockdown of unc-104 in IPCs impaired ILP secretion and reduced peripheral insulin signaling. Unc-104 appears to transport ILPs along axons. As a complementary approach, we tested dominant-negative Rab genes to find Rab proteins required in IPCs for ILP production or secretion. Rab1 was identified as crucial for ILP trafficking in IPCs. Inhibition of Rab1 in IPCs increased circulating sugar levels, delayed development, and lowered weight and body size. Immunofluorescence labeling of Rab1 showed its tight association with ILP2 in the Golgi of IPCs. Unc-104 and Rab1 join other proteins required for ILP transport in IPCs.
Glomerulonephritis was found to be the most common cause of ESRD in all the studied patients. The rural patients with ESRD in Guangxi had kidney disease in more advanced stage at the time of first visit to a nephrologist and higher rates of mortality after dialysis initiation, compared to urban patients.
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