Case report: Anti-ARHGAP26 autoantibodies in atypical dementia with Lewy bodies.

Background: Dementia with Lewy bodies (DLB) is the second most common type of neurodegenerative dementia. Here, we report a case of dementia associated with anti-Rho-GTPase-activating protein 26 (ARHGAP26) autoantibodies, which have never been previously linked to DLB. Methods: We describe the case of a 78-year-old man who underwent cerebrospinal fluid (CSF) analysis, magnetic resonance imaging (MRI), 18F-fluorodesoxyglucose positron emission tomography (FDG-PET), and a detailed neuropsychological evaluation. Results: The patient presented with mild dementia syndrome associated with extrapyramidal symptoms. Neuropsychological testing revealed impaired cognitive flexibility, figural memory, and verbal memory. Fluctuating cognitive abilities with deficits in attention-executive dysfunction and visuoconstruction also developed over time. A brain MRI showed reduced biparietal and cerebellar brain volume with generalized accentuation of the outer CSF spaces. The patient's CSF revealed anti-ARHGAP26 autoantibodies, which were also detectable in serum. In the differential complementary imaging diagnosis at 2 years, an FDG-PET revealed decreased occupancy of the posterior cingulum and precuneus. Although the FDG-PET, MRI, and clinical findings were potentially consistent with Alzheimer's disease, negative amyloid biomarkers in the CSF made an AD diagnosis highly unlikely. Single photon emission computed tomography (SPECT) with [(123)I] N-omega-fluoropropyl-2beta-carbomethoxy-3beta-{4-iodophenyl}nortropane ([(123)I]FP-CIT) showed right-sided predominance, reduced dopamine transporter uptake in the putamen, consistent with a positive indicative biomarker finding typical of DLB. Considering the clinically probable DLB associated with the two core features of Parkinsonism and fluctuating cognition with deficits in attention, supported by an abundant tracer uptake in the right putamen and lower uptake in the left putamen on 123I-FP-CIT-SPECT as an indicative biomarker, we started an antidementia drug using a cholinesterase inhibitor. Conclusions: Our report shows that atypical DLB may be associated with anti-ARHGAP26 autoantibodies, although their role and significance in the pathogenesis of DLB are unknown. However, it has to be mentioned that it is also possible that antibody-specific synthesis of anti-ARHGAP26 autoantibodies is a hallmark of a rare autoimmune disease that may cause the clinical and laboratory features involving altered dopamine transporter uptake on 123I-FP-CIT-SPECT, dementia, and mild Parkinson's symptoms rather than idiopathic DLB with only two core DLB features and inconsistent cognitive and imaging findings. Further research is needed to investigate the role of these autoantibodies in different dementias, particularly in DLB and mixed DLB-AD types.

Rho GTPase-activating protein 10 (ARHGAP10/GRAF2) is a novel autoantibody target in patients with autoimmune encephalitis.

BACKGROUND: In 2010, we described a novel immunoglobulin G (IgG) autoantibody (termed anti-Ca after the index case) targeting Rho GTPase-activating protein 26 (ARHGAP26, also termed GTPase regulator associated with focal adhesion kinase [GRAF], or oligophrenin-like protein 1 [OPHN1L]) in autoimmune cerebellar ataxia (ACA). Later, ARHGAP26-IgG/anti-Ca was reported in patients with limbic encephalitis/cognitive decline or peripheral neuropathy. In several of the reported cases, the syndrome was associated with cancer. ARHGAP10/GRAF2, which is expressed throughout the central nervous system, shares significant sequence homology with ARHGAP26/GRAF. Mutations in the ARHGAP10 gene have been linked to cognitive and psychiatric symptoms and schizophrenia. OBJECTIVE: To assess whether ARHGAP26-IgG/anti-Ca co-reacts with ARHGAP10. METHODS: Serological testing for ARHGAP10/GRAF2 autoantibodies by recombinant cell-based assays and isotype and IgG subclass analyses. RESULTS: 26/31 serum samples (84%) from 9/12 (75%) ARHGAP26-IgG/anti-Ca-positive patients and 4/6 ARHGAP26-IgG/anti-Ca-positive CSF samples from four patients were positive also for ARHGAP10-IgG. ARHGAP10-IgG (termed anti-Ca2) remained detectable in the long-term (up to 109 months) and belonged mainly to the complement-activating IgG1 subclass. Median ARHGAP26-IgG/anti-Ca and median ARHGAP10-IgG/anti-Ca2 serum titres were 1:3200 and 1:1000, respectively, with extraordinarily high titres in some samples (ARHGAP26-IgG/anti-Ca: up to 1:1000,000; ARHGAP10-IgG: up to 1:32,000). ARHGAP26/anti-Ca serum titres exceeded those of ARHGAP10-IgG in all samples but one. A subset of patients was positive also for ARHGAP10-IgM and ARHGAP10-IgA. CSF/serum ratios and antibody index calculation suggested intrathecal production of ARHGAP26-IgG/anti-Ca and anti-ARHGAP10. Of 101 control samples, 100 were completely negative for ARHGAP10-IgG; a single control sample bound weakly (1:10) to the ARHGAP10-transfected cells. CONCLUSIONS: We demonstrate that a substantial proportion of patients with ARHGAP26-IgG/anti-Ca-positive autoimmune encephalitis co-react with ARHGAP10. Further studies on the clinical and diagnostic implications of ARHGAP10-IgG/anti-Ca2 seropositivity in patients with autoimmune encephalitis are warranted.

Associations of ARHGAP26 Polymorphisms with Alzheimer's Disease and Cardiovascular Disease.

The Rho GTPase activating protein 26 (ARHGAP26) gene has been reported to be associated with neuropsychiatric diseases and neurodegenerative diseases including Parkinson's disease. We examined whether the ARHGAP26 gene is associated with Alzheimer's disease (AD) and/or cardiovascular disease (CVD). Multivariable logistic regression model was used to examine the associations of 154 single nucleotide polymorphisms (SNPs) within the ARHGAP26 gene with AD and CVD using the Alzheimer's Disease Neuroimaging Initiative 1 (ADNI-1) cohort. Fourteen SNPs were associated with AD (top SNP rs3776362 with p = 3.43 × 10-3), while 37 SNPs revealed associations with CVD (top SNP rs415235 with p = 2.06 × 10-4). Interestingly, 13 SNPs were associated with both AD and CVD. SNP rs3776362 was associated with CVD, Functional Activities Questionnaire (FAQ), and Clinical Dementia Rating Sum of Boxes (CDR-SB). A replication study using a Caribbean Hispanics sample showed that 17 SNPs revealed associations with AD, and 12 SNPs were associated with CVD. The third sample using a family-based study design showed that 9 SNPs were associated with AD, and 3 SNPs were associated with CVD. SNP rs6836509 within the ARHGAP10 gene (an important paralogon of ARHGAP26) was associated with AD and cerebrospinal fluid total tau (t-tau) level in the ADNI sample. Several SNPs were functionally important using the RegulomeDB, while a number of SNPs were associated with significant expression quantitative trait loci (eQTLs) using Genotype-Tissue Expression (GTEx) databases. In conclusion, genetic variants within ARHGAP26 were associated with AD and CVD. These findings add important new insights into the potentially shared pathogenesis of AD and CVD.

The role of GTPase-activating protein ARHGAP26 in human cancers.

Rho GTPases are molecular switches that play an important role in regulating the behavior of a variety of tumor cells. RhoA GTPase-activating protein 26 (ARHGAP26) is a GTPase-activating protein and inhibits the activity of Rho GTPases by promoting the hydrolytic ability of Rho GTPases. It also affects tumorigenesis and progression of various tumors through several methods, including formation of abnormal fusion genes and circular RNA. This review summarizes the biological functions and molecular mechanisms of ARHGAP26 in different tumors, proposes the potential clinical value of ARHGAP26 in cancer treatment, and discusses current issues that need to be addressed.

Deficiency of NEAT1 prevented MPP+-induced inflammatory response, oxidative stress and apoptosis in dopaminergic SK-N-SH neuroblastoma cells via miR-1277-5p/ARHGAP26 axis.

Noncoding RNAs including long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been documented to play prominent role in neurodegenerative diseases including Parkinson's disease (PD). This study intended to investigate the role of lncRNA nuclear enriched assembly transcript 1 (NEAT1) in MPP+-induced PD model in dopaminergic neuronblastoma SK-N-SH cells, as well as its mechanism through sponging miRNA (miR)-1277-5p. Real-time PCR and western blotting revealed that NEAT1 and ARHGAP26 were upregulated, and miR-1277-5p was downregulated in MPP+-treated SK-N-SH cells in a certain of concentration- and time- dependent manner. MPP+ induced apoptosis in SK-N-SH cells, as evidenced by decreased cell viability and Bcl-2 expression, and elevated apoptosis rate and levels of Bax and cleaved caspase-3, which were examined by MTT assay, flow cytometry and western blotting. Moreover, commercial assay kits indicated that inflammatory response and oxidative stress were provoked in response to MPP+, due to promoted contents of interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, malondialdehyde, and lactate dehydrogenase, accompanied with suppressed superoxide dismutase and glutathione peroxidase levels. Notably, MPP+-induced apoptosis, inflammatory response and oxidative stress in SK-N-SH cells were mitigated by NEAT1 knockdown and/or miR-1277-5p overexpression. Moreover, silencing of miR-1277-5p could abrogate the suppression of NEAT1 deficiency on MPP+-induced cell injury. Similarly, upregulating miR-1277-5p-elicited neuroprotection in MPP+-induced SK-N-SH cells was reversed by ARHGAP26 restoration. Dual-luciferase reporter assay demonstrated a direct interaction between miR-1277-5p and NEAT1 or ARHGAP26. Collectively, NEAT1 upregulation might contribute to MPP+-induced neuron injury via NEAT1-miR-1277-5p-ARHGAP26 competing endogenous RNAs (ceRNAs) pathway.

Circular RNA ARHGAP26 is over-expressed and its downregulation inhibits cell proliferation and promotes cell apoptosis in gastric cancer cells.

BACKGROUND/AIMS: This study aimed to explore the effect of circular RNA ARHGAP26 (circ-ARHGAP26) on cell proliferation and apoptosis in gastric cancer (GC) cell lines. MATERIALS AND METHODS: Human GC cell lines including HGC-27, AGS, SGC-7901, BGC-823, NCI-N87 and human normal gastric mucosal cells GSE-1 were cultured. The circ-ARHGAP26 expression was determined by quantitative polymerase chain reaction assay. Blank inhibitor and circ-ARHGAP26 inhibitor plasmids were transfected into HGC-27 or AGS cells as NC (-) and circ-ARHGAP26(-) groups. Counting Kit-8 (CCK-8) and Annexin V (AV)/propidium iodide (PI) were conducted to evaluate cell proliferation and cell apoptosis, respectively. Western blot was performed to determine the expressions of apoptotic markers (C-Caspase3 and Bcl-2). RESULTS: The circ-ARHGAP26 expression was elevated in HGC-27 (P < 0.001), AGS (P < 0.001), SGC-7901 (P < 0.01), BGC-823 (P < 0.05) and NCI-N87 (P < 0.05) GC cell lines compared to GSE-1 cells. In HGC-27 cells, CCK8 assay revealed that cell proliferation was decreased at 48 h (P < 0.05) and 72 h (P < 0.01), while AV/PI assay disclosed that cell apoptosis rate was increased at 72 h in circ-ARHGAP26 (-) group compared to NC (-) group (P < 0.01). Western blot assay also illuminated that apoptotic marker C-Caspase 3 was raised, while anti-apoptotic marker Bcl-2 was reduced at 72 h in circ-ARHGAP26 (-) group compared to NC (-) group. In addition, further validation in AGS cells also exhibited that cells proliferation was repressed, while apoptosis was enhanced in circ-ARHGAP26 (-) group compared to NC (-) group. CONCLUSION: The circ-ARHGAP26 is over-expressed and its downregulation inhibits cell proliferation and promotes cells apoptosis in GC cells.

RhoA/ROCK/ARHGAP26 signaling in the eutopic and ectopic endometrium is involved in clinical characteristics of adenomyosis.

OBJECTIVE: This study aimed to investigate RhoA, RhoA-associated coiled-coil containing protein kinase (ROCK) 1, ROCK2, and Rho GTPase-activating protein 26 (ARHGAP26) expression in the eutopic endometrium (EU) and ectopic endometrium (EC), and examine their relationships with the clinical characteristics of adenomyosis. METHODS: Twenty patients with adenomyosis who underwent laparoscopy were recruited. Protein and mRNA expression of RhoA, ROCK1, ROCK2, and ARHGAP26 in EU and EC of patients with adenomyosis and in control endometrium without adenomyosis (CE) was detected. RESULTS: ROCK1, ROCK2, and RhoA mRNA expression in EU was significantly higher than that in CE, and was highest in EC. ARHGAP26 mRNA expression in EC and EU was significantly lower than that in CE. ROCK1, ROCK2, and RhoA protein expression in EC and EU was significantly higher than that in CE. ARHGAP26 protein expression in EC and EU was significantly lower than that in CE. ROCK1, ROCK2, and RhoA gene and protein expression was positively associated and ARHGAP26 was negatively associated with the severity of menorrhagia and menstrual capacity in adenomyosis. CONCLUSIONS: RhoA, ROCK1, and ROCK2 expression is upregulated, and ARHGAP26 expression is downregulated in adenomyosis. The RhoA/ROCK-mediated signaling pathway is associated with dysmenorrhea and menstrual capacity in adenomyosis.

Anti-ARHGAP26 Autoantibodies Are Associated With Isolated Cognitive Impairment.

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.

TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators.

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.

Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion.

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.

From dizziness to severe ataxia and dysarthria: New cases of anti-Ca/ARHGAP26 autoantibody-associated cerebellar ataxia suggest a broad clinical spectrum.

In 2010, a novel anti-neuronal autoantibody, termed anti-Ca, was described in a patient with subacute cerebellar ataxia, and Rho GTPase-activating protein 26 (ARHGAP26) was identified as the target antigen. Recently, three additional cases of anti-Ca-positive cerebellar ataxia have been published. In addition to ataxia, cognitive decline and depression have been observed in some patients. Here, we report two new cases of anti-Ca-associated autoimmune cerebellar ataxia. Patient 1 presented with dizziness and acute yet mild limb and gait ataxia. Symptoms stabilized with long-term oral corticosteroid therapy but transiently worsened when steroids were tapered. Interestingly, both initial occurrence and worsening of the patient's neurological symptoms after steroid withdrawal were accompanied by spontaneous cutaneous hematomas. Patient 2 initially presented with an increased startle response and myoclonic jerks, and subsequently developed severe limb and gait ataxia, dysarthria, oculomotor disturbances, head and voice tremor, dysphagia, cognitive symptoms and depression. Steroid treatment was started five years after disease onset. The symptoms then responded only poorly to corticosteroids. At most recent follow-up, 19 years after disease onset, the patient was wheelchair-bound. These cases extend the clinical spectrum associated with anti-ARHGAP26 autoimmunity and suggest that early treatment may be important in patients with this rare syndrome.

Psychotic syndrome associated with anti-Ca/ARHGAP26 and voltage-gated potassium channel antibodies.

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. CASE REPORT: 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. CONCLUSION: 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.

GRAF1a is a brain-specific protein that promotes lipid droplet clustering and growth, and is enriched at lipid droplet junctions.

Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We found that GRAF1a, the longest GRAF1 isoform (GRAF1 is also known as ARHGAP26), was enriched in the brains of neonates. Endogenous GRAF1a was found on lipid droplets in oleic-acid-fed primary glial cells. Exclusive localization required a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promoted lipid droplet clustering, inhibited droplet mobility and severely perturbed lipolysis following the chase of cells overloaded with fatty acids. Under these conditions, GRAF1a concentrated at the interface between lipid droplets. Although GRAF1-knockout mice did not show any gross abnormal phenotype, the total lipid droplet volume that accumulated in GRAF1(-/-) primary glia upon incubation with fatty acids was reduced compared to GRAF1(+/+) cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis.

Anti-Ca/anti-ARHGAP26 antibodies associated with cerebellar atrophy and cognitive decline.

Recently, we identified a novel Purkinje cell-specific autoantibody (termed anti-Ca) targeting rhoGTPase-activating-protein-26 (ARHGAP26) in a patient with cerebellar ataxia. Here we describe a new case of anti-Ca/ARHGAP26 antibody-positive cerebellar ataxia. Cerebellar ataxia was associated with signs of possible limbic encephalitis in this case. The 24-year-old man presented with subacute pancerebellar ataxia, flattened affect, and cognitive decline. Neuropsychological testing revealed working memory deficits, compromised verbal learning and recall, attention deficits, slowed information processing, interference difficulty, and reduced spatial recognition. MRI showed severe pancerebellar atrophy. Serological examinations revealed high-titre anti-Ca/anti-ARHGAP26 antibodies. The antibodies belonged to the IgG1 subclass and were produced intrathecally. This case further corroborates the association of anti-Ca antibodies with cerebellar ataxia, expands the clinical spectrum, and highlights the necessity of antigen-specific diagnostic testing in immune-mediated cerebellar ataxia.

ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding.

Rho GTPase activating protein 26 (ARHGAP26) is a negative regulator of the Rho family that converts the small G proteins RhoA and Cdc42 to their inactive GDP-bound forms. It is essential for the CLIC/GEEC endocytic pathway, cell spreading, and muscle development. The present study shows that ARHGAP26 mRNA undergoes extensive A-to-I RNA editing in the 3' UTR that is specifically catalyzed by ADAR1. Furthermore, the mRNA and protein levels of ARHGAP26 were decreased in cells in which ADAR1 was knocked down. Conversely, ADAR1 overexpression increased the abundance of ARHGAP26 mRNA and protein. In addition, we found that both miR-30b-3p and miR-573 target the ARHGAP26 gene and that RNA editing of ARHGAP26 mediated by ADAR1 abolished the repression of its expression by miR-30b-3p or miR-573. When ADAR1 was overexpressed, the reduced abundance of ARHGAP26 protein mediated by miR-30b-3p or miR-573 was rescued. Importantly, we also found that knocking down ADAR1 elevated RhoA activity, which was consistent with the reduced level of ARHGAP26. Conversely, when ADAR1 was overexpressed, the amount of RhoA-GTP decreased. The similar expression patterns of ARHGAP26 and ADAR1 in human tissue samples further confirmed our findings. Taken together, our results suggest that ADAR1 regulates the expression of ARHGAP26 through A-to-I RNA editing by disrupting the binding of miR-30b-3p and miR-573 within the 3' UTR of ARHGAP26. This study provides a novel insight into the mechanism by which ADAR1 and its RNA editing function regulate microRNA-mediated modulation of target genes.