Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.

Identification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Mfn2 and Opa1.

Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier protein SLC25A46 interacts with both the outer and inner-membrane dynamin family GTPases Mfn1/2 and Opa1. While SLC25A46 levels are known affect mitochondrial morphology, how SLC25A46 interacts with Mfn1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass-spectrometry and AlphaFold 2 modeling to identify interfaces mediating a SLC25A46-Opa1-Mfn1/2 complex. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and the helical repeat 1 region of Mfn2 interacts with the SLC25A46 N-terminus. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance.

Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human Neurons.

ALS-linked mutations induce aberrant conformations within the SOD1 protein that are thought to underlie the pathogenic mechanism of SOD1-mediated ALS. Although clinical trials are underway for gene silencing of SOD1, these approaches reduce both wild-type and mutated forms of SOD1. Here, we sought to develop anti-SOD1 nanobodies with selectivity for mutant and misfolded forms of human SOD1 over wild-type SOD1. Characterization of two anti-SOD1 nanobodies revealed that these biologics stabilize mutant SOD1 in vitro. Further, SOD1 expression levels were enhanced and the physiological subcellular localization of mutant SOD1 was restored upon co-expression of anti-SOD1 nanobodies in immortalized cells. In human motor neurons harboring the SOD1 A4V mutation, anti-SOD1 nanobody expression promoted neurite outgrowth, demonstrating a protective effect of anti-SOD1 nanobodies in otherwise unhealthy cells. In vitro assays revealed that an anti-SOD1 nanobody exhibited selectivity for human mutant SOD1 over endogenous murine SOD1, thus supporting the preclinical utility of anti-SOD1 nanobodies for testing in animal models of ALS. In sum, the anti-SOD1 nanobodies developed and presented herein represent viable biologics for further preclinical testing in human and mouse models of ALS.

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

Absence of Cardiolipin From the Outer Leaflet of a Mitochondrial Inner Membrane Mimic Restricts Opa1-Mediated Fusion.

Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach.

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics.

Mitochondrial dynamics is essential for the organelle's diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Heterogeneity in human hippocampal CaMKII transcripts reveals allosteric hub-dependent regulation.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in Ca2+ signaling throughout the body. In the hippocampus, CaMKII is required for learning and memory. Vertebrate genomes encode four CaMKII homologs: CaMKIIα, CaMKIIß, CaMKIIγ, and CaMKIIδ. All CaMKIIs consist of a kinase domain, a regulatory segment, a variable linker region, and a hub domain, which is responsible for oligomerization. The four proteins differ primarily in linker length and composition because of extensive alternative splicing. Here, we report the heterogeneity of CaMKII transcripts in three complex samples of human hippocampus using deep sequencing. We showed that hippocampal cells contain a diverse collection of over 70 CaMKII transcripts from all four CaMKII-encoding genes. We characterized the Ca2+/CaM sensitivity of hippocampal CaMKII variants spanning a broad range of linker lengths and compositions. The effect of the variable linker on Ca2+/CaM sensitivity depended on the kinase and hub domains. Moreover, we revealed a previously uncharacterized role for the hub domain as an allosteric regulator of kinase activity, which may provide a pharmacological target for modulating CaMKII activity. Using small-angle x-ray scattering and single-particle cryo-electron microscopy (cryo-EM), we present evidence for extensive interactions between the kinase and the hub domains, even in the presence of a 30-residue linker. Together, these data suggest that Ca2+/CaM sensitivity in CaMKII is homolog dependent and includes substantial contributions from the hub domain. Our sequencing approach, combined with biochemistry, provides insights into understanding the complex pool of endogenous CaMKII splice variants.

Two forms of Opa1 cooperate to complete fusion of the mitochondrial inner-membrane.

Mitochondrial membrane dynamics is a cellular rheostat that relates metabolic function and organelle morphology. Using an in vitro reconstitution system, we describe a mechanism for how mitochondrial inner-membrane fusion is regulated by the ratio of two forms of Opa1. We found that the long-form of Opa1 (l-Opa1) is sufficient for membrane docking, hemifusion and low levels of content release. However, stoichiometric levels of the processed, short form of Opa1 (s-Opa1) work together with l-Opa1 to mediate efficient and fast membrane pore opening. Additionally, we found that excess levels of s-Opa1 inhibit fusion activity, as seen under conditions of altered proteostasis. These observations describe a mechanism for gating membrane fusion.

Modulation of actin polymerization affects nucleocytoplasmic transport in multiple forms of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.

Structural basis for mutation-induced destabilization of profilin 1 in ALS.

Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.