Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation

Abstract

Hundreds of L1CAM gene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditional L1CAM loss-of-function mutation and produced precisely matching control and L1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion of L1CAM dramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20–50% and ∼20–30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that the L1CAM mutation selectively decreased activity-dependent Na+-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations of L1CAM mutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.

ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Aβ Secretion

“…1A, 1B, S1A; Zhang et al, 2013). In previous studies (e.g., Pak et al, 2015; Patzke et al, 2016), we co-cultured human neurons with mouse glia because glia secretes multiple factors that support survival and synaptogenesis (Ullian et al, 2001). However, here we aimed to develop an approach that would allow examining the effects of glial factors on neurons, making it necessary to culture human neurons without glia.…”

“…Immunofluorescence staining experiments and image acquisition and analyses were performed essentially as described (Pak et al, 2015; Patzke et al, 2016). Briefly, cultured neurons were fixed in 4% paraformaldehyde, 4% sucrose in PBS, permeabilized with 0.2% Triton X-100 in PBS, and blocked with 5% goat serum in PBS.…”

Open Chromatin Profiling in hiPSC-Derived Neurons Prioritizes Functional Noncoding Psychiatric Risk Variants and Highlights Neurodevelopmental Loci

“…For downstream analysis of the functional effect of SZ risk variants, we elected to use the NEUROG2 -induced neuronal method, which can rapidly generate a large quantity of excitatory induced neurons (iNs) of high purity (Patzke et al, 2016; Zhang et al, 2013; Figure S5A). We obtained excitatory iNs expressing TUBB3 and VGLUT1 within 3–4 weeks (Figure 4D; Figure S5A).…”

The evolving role of ankyrin-B in cardiovascular disease

“…64 Recently, AnkB and AnkG were shown to be decreased in human neurons with deletion of L1CAM , a gene that has been associated with hydrocephalus and intellectual disability. 65 Furthermore, seizures have been shown to correlate with AnkB-associated cardiac channelopathies. 38, 66, 67 Initially discovered in 1993 and 1995, the 440kDa AnkB (AnkB-440) and 480kDa AnkG (AnkG-480) isoforms, also known as giant ankyrins, are a result of the addition of a single large exon.…”