Synapsin E-domain is essential for α-synuclein function.

The cytosolic proteins synucleins and synapsins are thought to play cooperative roles in regulating synaptic vesicle (SV) recycling, but mechanistic insight is lacking. Here, we identify the synapsin E-domain as an essential functional binding-partner of α-synuclein (α-syn). Synapsin E-domain allows α-syn functionality, binds to α-syn, and is necessary and sufficient for enabling effects of α-syn at synapses of cultured mouse hippocampal neurons. Together with previous studies implicating the E-domain in clustering SVs, our experiments advocate a cooperative role for these two proteins in maintaining physiologic SV clusters.

Serine-129 phosphorylation of α-synuclein is an activity-dependent trigger for physiologic protein-protein interactions and synaptic function.

Phosphorylation of α-synuclein at the serine-129 site (α-syn Ser129P) is an established pathologic hallmark of synucleinopathies and a therapeutic target. In physiologic states, only a fraction of α-syn is phosphorylated at this site, and most studies have focused on the pathologic roles of this post-translational modification. We found that unlike wild-type (WT) α-syn, which is widely expressed throughout the brain, the overall pattern of α-syn Ser129P is restricted, suggesting intrinsic regulation. Surprisingly, preventing Ser129P blocked activity-dependent synaptic attenuation by α-syn-thought to reflect its normal function. Exploring mechanisms, we found that neuronal activity augments Ser129P, which is a trigger for protein-protein interactions that are necessary for mediating α-syn function at the synapse. AlphaFold2-driven modeling and membrane-binding simulations suggest a scenario where Ser129P induces conformational changes that facilitate interactions with binding partners. Our experiments offer a new conceptual platform for investigating the role of Ser129 in synucleinopathies, with implications for drug development.

Synapsin E-domain is essential for α-synuclein function.

The cytosolic proteins synucleins and synapsins are thought to play cooperative roles in regulating synaptic vesicle (SV) recycling, but mechanistic insight is lacking. Here we identify the synapsin E-domain as an essential functional binding-partner of α-synuclein (α-syn). Synapsin E-domain allows α-syn functionality, binds to α-syn, and is necessary and sufficient for enabling effects of α-syn at the synapse. Together with previous studies implicating the E-domain in clustering SVs, our experiments advocate a cooperative role for these two proteins in maintaining physiologic SV clusters.

Genotype & phenotype in Lowe Syndrome: specific OCRL1 patient mutations differentially impact cellular phenotypes.

Lowe Syndrome (LS) is a lethal genetic disorder caused by mutations in the OCRL1 gene which encodes the lipid 5' phosphatase Ocrl1. Patients exhibit a characteristic triad of symptoms including eye, brain and kidney abnormalities with renal failure as the most common cause of premature death. Over 200 OCRL1 mutations have been identified in LS, but their specific impact on cellular processes is unknown. Despite observations of heterogeneity in patient symptom severity, there is little understanding of the correlation between genotype and its impact on phenotype. Here, we show that different mutations had diverse effects on protein localization and on triggering LS cellular phenotypes. In addition, some mutations affecting specific domains imparted unique characteristics to the resulting mutated protein. We also propose that certain mutations conformationally affect the 5'-phosphatase domain of the protein, resulting in loss of enzymatic activity and causing common and specific phenotypes (a conformational disease scenario). This study is the first to show the differential effect of patient 5'-phosphatase mutations on cellular phenotypes and introduces a conformational disease component in LS. This work provides a framework that explains symptom heterogeneity and can help stratify patients as well as to produce a more accurate prognosis depending on the nature and location of the mutation within the OCRL1 gene.

Lowe syndrome patient cells display mTOR- and RhoGTPase-dependent phenotypes alleviated by rapamycin and statins.

Lowe syndrome (LS) is an X-linked developmental disease characterized by cognitive deficiencies, bilateral congenital cataracts and renal dysfunction. Unfortunately, this disease leads to the early death of affected children often due to kidney failure. Although this condition was first described in the early 1950s and the affected gene (OCRL1) was identified in the early 1990s, its pathophysiological mechanism is not fully understood and there is no LS-specific cure available to patients. Here we report two important signaling pathways affected in LS patient cells. While RhoGTPase signaling abnormalities led to adhesion and spreading defects as compared to normal controls, PI3K/mTOR hyperactivation interfered with primary cilia assembly (scenario also observed in other ciliopathies with compromised kidney function). Importantly, we identified two FDA-approved drugs able to ameliorate these phenotypes. Specifically, statins mitigated adhesion and spreading abnormalities while rapamycin facilitated ciliogenesis in LS patient cells. However, no single drug was able to alleviate both phenotypes. Based on these and other observations, we speculate that Ocrl1 has dual, independent functions supporting proper RhoGTPase and PI3K/mTOR signaling. Therefore, this study suggest that Ocrl1-deficiency leads to signaling defects likely to require combinatorial drug treatment to suppress patient phenotypes and symptoms.

A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF-anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.

Cellular clearance of circulating transthyretin decreases cell-nonautonomous proteotoxicity in Caenorhabditis elegans.

Cell-autonomous and cell-nonautonomous mechanisms of neurodegeneration appear to occur in the proteinopathies, including Alzheimer's and Parkinson's diseases. However, how neuronal toxicity is generated from misfolding-prone proteins secreted by nonneuronal tissues and whether modulating protein aggregate levels at distal locales affects the degeneration of postmitotic neurons remains unknown. We generated and characterized animal models of the transthyretin (TTR) amyloidoses that faithfully recapitulate cell-nonautonomous neuronal proteotoxicity by expressing human TTR in the Caenorhabditis elegans muscle. We identified sensory neurons with affected morphological and behavioral nociception-sensing impairments. Nonnative TTR oligomer load and neurotoxicity increased following inhibition of TTR degradation in distal macrophage-like nonaffected cells. Moreover, reducing TTR levels by RNAi or by kinetically stabilizing natively folded TTR pharmacologically decreased TTR aggregate load and attenuated neuronal dysfunction. These findings reveal a critical role for in trans modulation of aggregation-prone degradation that directly affects postmitotic tissue degeneration observed in the proteinopathies.

Role of Ocrl1 in primary cilia assembly.

Lowe syndrome is a lethal X-linked genetic disorder characterized by congenital cataracts, mental retardation, and kidney dysfunction. It is caused by mutations in the OCRL1 (oculocerebrorenal syndrome of Lowe) gene that encodes a phosphatidylinositol 5-phosphatase (EC 3.1.3.36). The gene product Ocrl1 has been linked to a multitude of functions due to the central role played by phosphoinositides in signaling. Moreover, this protein also has the ability to bind Rho GTPases, the master regulators of the actin cytoskeleton, and to interact with elements of the vesicle trafficking machinery. It is currently under investigation how deficiencies in Ocrl1 affect these different processes and contribute to patient symptoms. This chapter outlines the known physiological roles of Ocrl1 which might be relevant to the mechanism underlying Lowe syndrome.

Ciliopathies: the trafficking connection.

The primary cilium (PC) is a very dynamic hair-like membrane structure that assembles/disassembles in a cell-cycle-dependent manner and is present in almost every cell type. Despite being continuous with the plasma membrane, a diffusion barrier located at the ciliary base confers the PC properties of a separate organelle with very specific characteristics and membrane composition. Therefore, vesicle trafficking is the major process by which components are acquired for cilium formation and maintenance. In fact, a system of specific sorting signals controls the right of cargo admission into the cilia. Disruption to the ciliary structure or its function leads to multiorgan diseases known as ciliopathies. These illnesses arise from a spectrum of mutations in any of the more than 50 loci linked to these conditions. Therefore, it is not surprising that symptom variability (specific manifestations and severity) among and within ciliopathies appears to be an emerging characteristic. Nevertheless, one can speculate that mutations occurring in genes whose products contribute to the overall vesicle trafficking to the PC (i.e. affecting cilia assembly) will lead to more severe symptoms, whereas those involved in the transport of specific cargoes will result in milder phenotypes. In this review, we summarize the trafficking mechanisms to the cilia and also provide a description of the trafficking defects observed in some ciliopathies which can be correlated to the severity of the pathology.

The epsin protein family: coordinators of endocytosis and signaling.

The epsins are a conserved family of endocytic adaptors essential for cell viability in yeast and for embryo development in higher eukaryotes. Epsins function as adaptors by recognizing ubiquitinated cargo and as endocytic accessory proteins by contributing to endocytic network stability/regulation and membrane bending. Importantly, epsins play a critical role in signaling by contributing to epidermal growth factor receptor downregulation and the activation of notch and RhoGTPase pathways. In this review, we present an overview of the epsins and emphasize their functional importance as coordinators of endocytosis and signaling.

The Lowe syndrome protein OCRL1 is involved in primary cilia assembly.

Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.

Lowe syndrome: Between primary cilia assembly and Rac1-mediated membrane remodeling.

Lowe syndrome (LS) is a lethal X-linked genetic disease caused by functional deficiencies of the phosphatidlyinositol 5-phosphatase, Ocrl1. In the past four years, our lab described the first Ocrl1-specific cellular phenotypes using dermal fibroblasts from LS patients. These phenotypes, validated in an ocrl1-morphant zebrafish model, included membrane remodeling (cell migration/spreading, fluid-phase uptake) defects and primary cilia assembly abnormalities. On one hand, our findings unraveled cellular phenotypes likely to be involved in the observed developmental defects; on the other hand, these discoveries established LS as a ciliopathy-associated disease. This article discusses the possible mechanisms by which loss of Ocrl1 function may affect RhoGTPase signaling pathways leading to actin cytoskeleton rearrangements that underlie the observed cellular phenotypes.