The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, PRKN/Parkin facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this pathway contribute to the pathogenesis of numerous cardiometabolic and neurological diseases. Although dynamic actin remodeling has recently been shown to play an important role in governing spatiotemporal control of mitophagy, the mechanisms remain unclear. We recently found that the RhoGAP, ARHGAP26/GRAF1 is a PRKN-binding protein that is rapidly recruited to damaged mitochondria where upon phosphorylation by PINK1 it serves to coordinate phagophore capture by regulating mitochondrial-associated actin remodeling and by facilitating PRKN-LC3 interactions. Because ARHGAP26 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure and ARHGAP26 depletion in mouse hearts blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress, this enzyme may be a tractable target to treat the many diseases associated with mitochondrial dysfunction.

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.

Adipose tissue, which is crucial for the regulation of energy within the body, contains both white and brown adipocytes. White adipose tissue (WAT) primarily stores energy, while brown adipose tissue (BAT) plays a critical role in energy dissipation as heat, offering potential for therapies aimed at enhancing metabolic health. Regulation of the RhoA/ROCK pathway is crucial for appropriate specification, differentiation and maturation of both white and brown adipocytes. However, our knowledge of how this pathway is controlled within specific adipose depots remains unclear, and to date a RhoA regulator that selectively controls adipocyte browning has not been identified. Our study shows that expression of GRAF1, a RhoGAP highly expressed in metabolically active tissues, closely correlates with brown adipocyte differentiation in culture and in vivo. Mice with either global or adipocyte-specific GRAF1 deficiency exhibit impaired BAT maturation, reduced capacity for WAT browning, and compromised cold-induced thermogenesis. Moreover, defects in differentiation of mouse or human GRAF1-deficient brown preadipocytes can be rescued by treatment with a Rho kinase inhibitor. Collectively, these studies indicate that GRAF1 can selectively induce brown and beige adipocyte differentiation and suggest that manipulating GRAF1 activity may hold promise for the future treatment of diseases related to metabolic dysfunction.

: T cell activation is immediately followed by internalization of the T cell receptor (TCR). TCR endocytosis is required for T cell activation, but the mechanisms supporting removal of TCR from the cell surface remain incompletely understood. Here we report that TCR endocytosis is linked to the clathrin-independent carrier (CLIC) and GPI-enriched endocytic compartments (GEEC) endocytic pathway. We show that unlike the canonical clathrin cargo transferrin or the adaptor protein Lat, internalized TCR accumulates in tubules shaped by the small GTPase Cdc42 and the Bin/amphiphysin/Rvs (BAR) domain containing protein GRAF1 in T cells. Preventing GRAF1-positive tubules to mature into endocytic vesicles by expressing a constitutively active Cdc42 impairs the endocytosis of TCR, while having no consequence on the uptake of transferrin. Together, our data reveal a link between TCR internalization and the CLIC/GEEC endocytic route supported by Cdc42 and GRAF1.

The intracellular tyrosine kinase Pyk2 (PTK2B) is related to focal adhesion kinase and localizes to postsynaptic sites in brain. Pyk2 genetic variation contributes to late onset Alzheimer\'s disease (AD) risk. We recently observed that Pyk2 is required for synapse loss and for learning deficits in a transgenic mouse model of AD. Here, we explore the cellular and biochemical basis for the action of Pyk2 tyrosine kinase in amyloid-β oligomer (Aβo)-induced dendritic spine loss. Overexpression of Pyk2 reduces dendritic spine density of hippocampal neurons by a kinase-dependent mechanism. Biochemical isolation of Pyk2-interacting proteins from brain identifies Graf1c, a RhoA GTPase-activating protein inhibited by Pyk2. Aβo-induced reductions in dendritic spine motility and chronic spine loss require both Pyk2 kinase and RhoA activation. Thus, Pyk2 functions at postsynaptic sites to modulate F-actin control by RhoA and regulate synapse maintenance of relevance to AD risk.SIGNIFICANCE STATEMENT Genetic variation at the Pyk2 locus is a risk for Alzheimer\'s disease. We have observed that Pyk2 is required for AD transgenic synapse loss and memory dysfunction. However, the cellular and biochemical basis for Pyk2 function related to AD is not defined. Here, we show that brain Pyk2 interacts with the RhoGAP protein Graf1 to alter dendritic spine stability via RhoA GTPase. Amyloid-β oligomer-induced dendritic spine loss requires the Pyk2/Graf1 pathway.

Autoantibodies against the RhoGTPase-activating protein 26 (ARHGAP26) were originally identified in the context of subacute autoimmune cerebellar ataxia. Further studies identified a wider clinical spectrum including psychotic, affective, and cognitive symptoms. Only a few patients reported so far had evidence of a tumor association. A prospective analysis between January 2015 and December 2017 at the Dept. of Neurology at Charité-Universitätsmedizin Berlin identified 14 patients with ARHGAP26 autoantibodies on a cell-based assay, of which three patients had additional brain immunohistochemistry staining of cerebellar molecular layer and Purkinje cells, who were therefore considered antibody-positive. In all three patients, ARHGAP26 autoantibodies were associated with tumors. In two patients, an isolated cognitive impairment without additional neurological deficits was observed. These cases thus further extend the clinical spectrum associated with ARHGAP26 autoantibodies and strengthen a potential paraneoplastic context.

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

Adaptation of cell shape and polarization through the formation and retraction of cellular protrusions requires balancing of endocytosis and exocytosis combined with fine-tuning of the local activity of small GTPases like Rab8. Here, we show that endocytic turnover of the plasma membrane at protrusions is directly coupled to surface removal and inactivation of Rab8. Removal is induced by reduced membrane tension and mediated by the GTPase regulator associated with focal adhesion kinase-1 (GRAF1, also known as ARHGAP26), a regulator of clathrin-independent endocytosis. GRAF1-depleted cells were deficient in multi-directional spreading and displayed elevated levels of GTP-loaded Rab8, which was accumulated at the tips of static protrusions. Furthermore, GRAF1 depletion impaired lumen formation and spindle orientation in a 3D cell culture system, indicating that GRAF1 activity regulates polarity establishment. Our data suggest that GRAF1-mediated removal of Rab8 from the cell surface restricts its activity during protrusion formation, thereby facilitating dynamic adjustment of the polarity axis.

Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase-activating protein (GAP) GRAF1 (also known as ARHGAP26), facilitates rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid-phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, which were distinct from clathrin-coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane, resulting in a corresponding decrease in the microdomain association of Cdc42. However, Cdc42 captured in its active state was, through a GAP-domain-mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism.

BACKGROUND: Antibodies to the Rho GTPase-activating protein 26 (ARHGAP26, GRAF1) (also termed anti-Ca) were first described in patients with cerebellar ataxia. However, ARHGAP26 is also expressed in some hippocampal neurons. Moreover, some of the previously reported patients showed cognitive and affective symptoms. It is unknown whether those symptoms reflected involvement of the limbic system or were part of the so-called cerebellar cognitive/affective syndrome.
METHODS: Here, we report a newly diagnosed anti-Ca/ARHGAP26-IgG-positive patient who presented with recurrent psychotic symptoms but no cerebellar ataxia. In addition, low-titer acetylcholine receptor antibodies, voltage-gated potassium channel complex antibodies (but no LGI1 or CASPR2 antibodies) and anti-nuclear antibodies of unknown specificity were detected, suggesting a general autoimmune predisposition. Thymectomy revealed mild thymic nodular hyperplasia.
CONCLUSIONS: This case indicates that the clinical spectrum of ARHGAP26-related autoimmunity might be broader than expected. Studies on the prevalence of anti-Ca/ARHGAP26 in patients with suspected limbic encephalitis seem warranted.