SYNGR4 and PLEKHB1 deregulation in motor neurons of amyotrophic lateral sclerosis models: potential contributions to pathobiology

Abstract

Amyotrophic lateral sclerosis is the most common adult-onset neurodegenerative disease affecting motor neurons. Its defining feature is progressive loss of motor neuron function in the cortex, brainstem, and spinal cord, leading to paralysis and death. Despite major advances in identifying genes that can cause disease when mutated and model the disease in animals and cellular models, it still remains unclear why motor symptoms suddenly appear after a long pre-symptomatic phase of apparently normal function. One hypothesis is that age-related deregulation of specific proteins within key cell types, especially motor neurons themselves, initiates disease symptom appearance and may also drive progressive degeneration. Genome-wide in vivo cell-type-specific screening tools are enabling identification of candidates for such proteins. In this minireview, we first briefly discuss the methodology used in a recent study that applied a motor neuron-specific RNA-Seq screening approach to a standard model of TAR DNA-binding protein-43 (TDP-43)-driven amyotrophic lateral sclerosis. A key finding of this study is that synaptogyrin-4 and pleckstrin homology domain-containing family B member 1 are also deregulated at the protein level within motor neurons of two unrelated mouse models of mutant TDP-43 driven amyotrophic lateral sclerosis. Guided by what is known about molecular and cellular functions of these proteins and their orthologs, we outline here specific hypotheses for how changes in their levels might potentially alter cellular physiology of motor neurons and detrimentally affect motor neuron function. Where possible, we also discuss how this information could potentially be used in a translational context to develop new therapeutic strategies for this currently incurable, devastating disease.

Keywords: TAR DNA-binding protein-43; amyotrophic lateral sclerosis; glucagon-like peptide-1 receptor; motor neuron disease; mouse model; neurodegeneration; phosphatidylserine; pleckstrin homology domain; synaptogyrin; vesicle transport.

Figure 1

Translatome screening reveals specific proteins deregulated within motor neurons (MNs) at symptom onset in mouse models of mutant TDP-43-driven amyotrophic lateral sclerosis (ALS). An overview of the experimental strategy used to screen for proteins that are deregulated within MNs at symptom onset in mutant TDP-43-driven amyotrophic lateral sclerosis. Spinal cords from mice of three different genotypes: Chat bacTRAP, Chat bacTRAP; hTDP-43^A315T, and Chat bacTRAP; hTDP-43^WT were extracted and lysed. MN ribosomes and associated mRNAs were purified via translating ribosome affinity purification (TRAP) and mRNAs were processed for RNA sequencing. This generated gene level datasets of specifically translated mRNAs in MNs (the “translatome”) which were analyzed for differential expression. Two candidate genes found to be exclusively deregulated in the mutant cohort were further validated at both the RNA and protein levels using qRT-PCR and tissue immunostaining, respectively. Collectively, this approach revealed upregulation of SYNGR4 and downregulation of PLEKHB1 within spinal MNs coincident with the onset of motor symptoms in mouse ALS models based on patient mutations in TDP-43 (Marques et al., 2020).

Figure 2

Possible cellular and pathobiological effects of altered SYNGR4 and PLEKHB1 protein levels in motor neurons (MNs). (A) By analogy to SYNGR2/cellugyrin, we propose the existence of intracellular SYNGR4 vesicles (S4Vs) engaged in storage and/or retrograde transport between endosomal compartments and the Trans-Golgi network (TGN) of specific cell-surface cargo proteins. Increased SYNGR4 leads to increased intracellular retention of specific S4V cargos, reducing their cell-surface levels, as shown for the candidate S4V cargo, GLP-1R, a key regulator of neuroprotective central nervous system insulin signaling. (B) Assuming PLEKHB1 plays a similar role to evectin-2 (evt-2)/PLEKHB2 in sorting of specific endosomal cargos for recycling to the TGN, reducing PLEKHB1 would shift some TGN proteins to the MN cell surface, potentially affecting relative levels of other proteins there as well. Additionally, two potential direct effects of reducing PLEKHB1 levels at the MN plasma membrane are shown. First, reduced PLEKHB1 binding to beta gamma subunits of yet-to-be-identified MN G-protein coupled receptors (GPCRs) could directly affect MN GPCR signaling. Second, reduced PLEKHB1 binding to phosphatidylserine (PS) could enable better access for scramblases, leading to increased PS on the outer leaflet of the MN membrane to serve as an “eat me” signal. In principle, any of these effects might also involve the direct physical interaction of PLEKHB1 with myosin 1c/VIIa motors. Note that while MN soma are shown for simplicity, synaptic and axonal effects are also possible. Since effects shown in A and B presumably occur simultaneously, future studies should consider potential interaction points (e.g. overlap in endosomal cargo proteins; potential PLEKHB1 interaction with GLP-1R, a GPCR). Red arrows reflect specific cellular pathways hypothesized to be affected by upregulation of SYNGR4 (A) and downregulation PLEKHB1 (B).