Biology subjects

Morrow, E. M.

Publications and source records attributed to Morrow, E. M..

8 recordsLinked to original sources

bioRxiv2026

Longitudinal single-cell transcriptional profiling identifies dynamic neurodegenerative mechanisms in Christianson Syndrome rat

Endolysosomal dysfunction is a central feature of many neurodegenerative disorders. Loss-of-function mutations in the endosomal Na+/H+ exchanger NHE6 cause lysosomal dysfunction and the early-onset neurodegenerative disorder Christianson syndrome (CS). CS shares pathological features with more common neurodegenerative diseases; however, the extent of mechanistic overlap remains unclear. Using single-nucleus RNA sequencing across the lifespan in a CS rat model, we define the temporal progression of disease at single-cell resolution in the hippocampus. Neurons exhibit early transcriptional signatures of mitochondrial dysfunction. We investigated mitochondrial function in vitro and ex vivo and discover progressive defects in mitochondrial quality control and bioenergetics in NHE6-null tissue. Furthermore, both transcriptomic and proteomic analyses reveal robust activation of innate immunity, including cGAS-STING signaling which localizes to microglia. Therefore, in CS rat brain, early neuronal endosomal and mitochondrial impairments are followed by pronounced microglial and astrocytic activation, as well as age-dependent decline in oligodendrocyte myelinating capacity. Cross species comparison reveals substantial overlap between single-cell analyses in CS rat and in human postmortem Alzheimers disease hippocampus. Together, our findings define the cellular trajectories of neurodegeneration in CS, and identify convergent endolysosomal, mitochondrial, and inflammatory mechanisms that support a broader role for endolysosomal dysfunction in Alzheimers disease pathogenesis.

neuroscience↗

bioRxiv2025

A transcriptional biosensor reveals mechanisms of α-ketoglutarate signaling to chromatin

Alpha-ketoglutarate (KG) is required for chromatin demethylation but mechanisms controlling KG abundance in the nucleus are poorly defined. Therefore, we designed a biosensor system to monitor this metabolite pool in human cells using an KG-responsive cyanobacterial transcription factor, NtcA. We then coupled this system with a genetic screen to identify genes that regulate KG in the nucleus, defining an inter-organelle pathway in which sequential mitochondrial activities of the GPT2 transaminase and SLC25A11 transporter supply nuclear KG. Using a mouse model of GPT2 deficiency, a human inborn error of metabolism, we found that this pathway controls chromatin methylation in the developing brain. Our work provides a tool to assess KG signaling to chromatin and a framework for leveraging forward genetics to study nuclear metabolite pools.

synthetic biology↗

bioRxiv2024

Endosome maturation is orchestrated by inside-out proton signaling through a Na+/H+ exchanger and pH-dependent Rab GTPase cycling

Endosome maturation requires progressive lumen acidification. To what extent is lumen acidification sensed by cytosolic-side molecules that drive endosome maturation? We show here that "inside-out" proton signaling through the endosomal Na+/H+ Exchanger 6 (NHE6) activates the late endosome master regulator Rab7. The mechanism involves potent inactivation of the Rab7 GTPase-activating protein (GAP) TBC1D5 with decreasing pH. NHE6 interacts with TBC1D5 in a complex with Rab7. Neurons from NHE6-null mice or mice engineered with a selective defect in NHE6 proton efflux exhibit blocked endosome maturation and decreased active Rab7, consistent with an overactive Rab7 GAP. Finally, epistatic knock-down of TBC1D5, thereby reducing Rab7 GAP activity, in NHE6-null neurons rescues Rab7 GTPase cycling and endosome maturation. Importantly, NHE6 is mutated in Christianson Syndrome underscoring the significance of these mechanisms to neurodegeneration. We conclude that lumen acidification regulates pH-dependent Rab GTPase cycling to coordinate late endosome maturation by a process involving proton signaling.

cell biology↗

bioRxiv2024

Loss of mitochondrial enzyme GPT2 leads to reprogramming of synaptic glutamate metabolism

Recessive loss-of-function mutations in the mitochondrial enzyme Glutamate Pyruvate Transaminase 2 (GPT2) cause intellectual disability in children. Given this cognitive disorder, and because glutamate metabolism is tightly regulated to sustain excitatory neurotransmission, here we investigate the role of GPT2 in synaptic function. GPT2 catalyzes a reversible reaction interconverting glutamate and pyruvate with alanine and alpha-ketoglutarate, a TCA cycle intermediate; thereby, GPT2 may play an important role in linking mitochondrial tricarboxylic acid (TCA) cycle with synaptic transmission. In mouse brain, we find that GPT2 is enriched in mitochondria of synaptosomes (isolated synaptic terminals). Loss of Gpt2 in mouse appears to lead to reprogramming of glutamate and glutamine metabolism, and to decreased glutamatergic synaptic transmission. Whole-cell patch-clamp recordings in pyramidal neurons of CA1 hippocampal slices from Gpt2-null mice reveal decreased excitatory post-synaptic currents (mEPSCs) without changes in mEPSC frequency, or importantly, changes in inhibitory post-synaptic currents (mIPSCs). Additional evidence of defective glutamate release included reduced levels of glutamate released from Gpt2-null synaptosomes measured biochemically. Glutamate release from synaptosomes was rescued to wild-type levels by alpha-ketoglutarate supplementation. Additionally, we observed evidence of altered metabolism in isolated Gpt2-null synaptosomes: decreased TCA cycle intermediates, and increased glutamate dehydrogenase activity. Notably, alterations in the TCA cycle and the glutamine pool were alleviated by alpha-ketoglutarate supplementation. In conclusion, our data support a model whereby GPT2 mitochondrial activity may contribute to glutamate availability in pre-synaptic terminals, thereby highlighting potential interactions between pre-synaptic mitochondrial metabolism and synaptic transmission.

neuroscience↗

bioRxiv2023

GGA1 interacts with the endosomal Na+/H+ Exchanger NHE6 governing localization to the endosome compartment

Mutations in the endosomal Na+/H+ exchanger (NHE6) cause Christianson syndrome (CS), an X-linked neurological disorder. Previous studies have shown that NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl-terminus as bait, we identify Golgi-associated, Gamma adaptin ear containing, ARF binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using co-immunoprecipitation (co-IP): using over-expressed constructs in mammalian cells; and co-IP of endogenously-expressed GGA1 and NHE6 from neuroblastoma cells, as well as from mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7 and NHE9) but not with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate that the cytoplasmic tail of NHE6 is necessary and sufficient for interactions with GGA1. We demonstrate the co-localization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mis-localized in GGA1 knockout cells wherein we find less NHE6 in endosomes but more NHE6 transport to lysosomes, and more Golgi retention of NHE6 with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mis-localization, and Golgi retention, we find the intra-luminal pH in Golgi to be alkalinized. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intra-cellular NHE to the endosome compartment.

cell biology↗

bioRxiv2021

Correction of a pathogenic mutation in iPSCs derived from a patient with Christianson syndrome using CRISPR/Cas9 genome editing

SLC9A6 (also termed NHE6) encodes the endosomal Na+/H+ exchanger 6 (NHE6). Pathogenic, loss-of-function mutations in NHE6 cause the X-linked neurogenetic disorder Christianson syndrome (CS). We developed induced pluripotent stem cell (iPSC) lines derived from a patient with CS and from a biologically related control. The patient with CS contained the nonsense mutation c.1569G>A (p.(W523X)), which caused a significant reduction in NHE6 mRNA and a lack of detectable NHE6 protein in CS iPSCs in comparison to control iPSCs. To establish a cell model for study of CS with an isogenic control, we corrected the c.1569G>A mutation to the NHE6 reference genome sequence using CRISPR/Cas9-mediated homology directed repair knock-in methodology. Multiple subclonal lines were generated, and notably, NHE6 protein was expressed in all analyzed c.1569G>A (p.(W523X)) genome-corrected iPSC lines. This CS iPSC model together with the associated biologically related and isogenic control cell lines will serve as a valuable resource for both basic and translational studies in CS.

genetics↗

bioRxiv2021

Generation of Pathogenic TPP1 Mutations in Human Stem Cells as a Model for CLN2 Disease

Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is an autosomal recessive neurodegenerative disorder generally with onset at 2 to 4 years of age and characterized by seizures, loss of vision, progressive motor and mental decline, and premature death. CLN2 disease is caused by loss-of-function mutations in the tripeptidyl peptidase 1 (TPP1) gene leading to deficiency in TPP1 enzyme activity. Approximately 60% of patients have one of two pathogenic variants (c.509-1G>C or c.622C>T [p.(Arg208*)]). In order to generate a human stem cell model of CLN2 disease, we used CRISPR/Cas9-mediated knock-in technology to introduce these mutations in a homozygous state into H9 human embryonic stem cells. Heterozygous lines of the c.622C>T (p.(Arg208*)) mutation were also generated, which included a heterozygous mutant with a wild-type allele and different compound heterozygous coding mutants resulting from indels on one allele. We describe the methodology that led to the generation of the lines and provide data on the initial validation and characterization of these CLN2 disease models. Notably, both mutant lines (c.509-1G>C and c.622C>T [p.(Arg208*)]) in the homozygous state were shown to have reduced or absent protein, respectively, and deficiency of TPP1 enzyme activity. These models, which we are making available for wide-spread sharing, will be useful for future studies of molecular and cellular mechanisms underlying CLN2 disease and for therapeutic development.

cell biology↗

bioRxiv2018

Human neurons from Christianson syndrome iPSCs reveal allele-specific responses to rescue strategies

Human genetic disorders provide a powerful lens to understanding the human brain. Induced pluripotent stem cells (iPSC) represent an important, new resource for mechanistic studies and therapeutic development. Christianson syndrome (CS), an X-linked neurological disorder with attenuation of brain growth postnatally (postnatal microcephaly), is caused by mutations in SLC9A6, the gene encoding endosomal Na+/H+ exchanger 6 (NHE6). We developed CS iPSC lines from patients with a mutational spectrum, as well as robust biologically-related and isogenic controls. We demonstrate that mutations in CS lead to loss of protein function by a variety of mechanisms. Regardless of mutation, all patient-derived neurons demonstrate reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Additionally, phenotype rescue strategies show allele-specific responses: a gene replacement strategy shows efficacy in nonsense mutations but not in a missense mutation, whereas application of exogenous trophic factors (BDNF or IGF-1) rescues arborization phenotypes across all mutations. Our data emphasize the important principle of personalized medicine whereby success of some therapeutic strategies may be more linked to patient genotype than others.

neuroscience↗