Proteostatic Remodeling of Small Heat Shock Chaperones─Crystallins by Ran-Binding Protein 2─and the Peptidyl-Prolyl cis-trans Isomerase and Chaperone Activities of Its Cyclophilin Domain.

Disturbances in protein phase transitions promote protein aggregation─a neurodegeneration hallmark. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also regulate phase transitions and proteostasis by suppressing protein aggregation. Ranbp2 haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against phototoxicity by proteostatic regulations of neuroprotective substrates of Ranbp2 and by suppressing the buildup of polyubiquitylated substrates. Losses of peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 recapitulate molecular effects of Ranbp2 haploinsufficiency. These CY impairments also stimulate deubiquitylation activities and phase transitions of 19S cap subunits of the 26S proteasome that associates with Ranbp2. However, links between CY moonlighting activity, substrate ubiquitylation, and proteostasis remain incomplete. Here, we reveal the Ranbp2 regulation of small heat shock chaperones─crystallins in the chorioretina by proteomics of mice with total or selective modular deficits of Ranbp2. Specifically, loss of CY PPIase of Ranbp2 upregulates αA-Crystallin, which is repressed in adult nonlenticular tissues. Conversely, impairment of CY's chaperone activity opposite to the PPIase pocket downregulates a subset of αA-Crystallin's substrates, γ-crystallins. These CY-dependent effects cause age-dependent and chorioretinal-selective declines of ubiquitylated substrates without affecting the chorioretinal morphology. A model emerges whereby inhibition of Ranbp2's CY PPIase remodels crystallins' expressions, subdues molecular aging, and preordains the chorioretina to neuroprotection by augmenting the chaperone capacity and the degradation of polyubiquitylated substrates against proteostatic impairments. Further, the druggable Ranbp2 CY holds pan-therapeutic potential against proteotoxicity and neurodegeneration.

Proteostatic remodeling of small heat shock chaperones - crystallins by Ran-binding protein 2 and the peptidyl-prolyl cis-trans isomerase and chaperone activities of its cyclophilin domain.

Disturbances in phase transitions and intracellular partitions of nucleocytoplasmic shuttling substrates promote protein aggregation - a hallmark of neurodegenerative diseases. The modular Ran-binding protein 2 (Ranbp2) is a cytosolic molecular hub for rate-limiting steps of disassembly and phase transitions of Ran-GTP-bound protein ensembles exiting nuclear pores. Chaperones also play central roles in phase transitions and proteostasis by suppressing protein aggregation. Ranbp2 haploinsufficiency promotes the age-dependent neuroprotection of the chorioretina against photo-oxidative stress by proteostatic regulations of Ranbp2 substrates and by countering the build-up of poly-ubiquitylated substrates. Further, the peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities of the cyclophilin domain (CY) of Ranbp2 modulate the proteostasis of selective neuroprotective substrates, such as hnRNPA2B1, STAT3, HDAC4 or L/M-opsin, while promoting a decline of ubiquitylated substrates. However, links between CY PPIase activity on client substrates and its effect(s) on ubiquitylated substrates are unclear. Here, proteomics of genetically modified mice with deficits of Ranbp2 uncovered the regulation of the small heat shock chaperones - crystallins by Ranbp2 in the chorioretina. Loss of CY PPIase of Ranbp2 up-regulates αA-crystallin proteostasis, which is repressed in non-lenticular tissues. Conversely, the αA-crystallin's substrates, γ-crystallins, are down-regulated by impairment of CY's C-terminal chaperone activity. These CY-dependent effects cause the age-dependent decline of ubiquitylated substrates without overt chorioretinal morphological changes. A model emerges whereby the Ranbp2 CY-dependent remodeling of crystallins' proteostasis subdues molecular aging and preordains chorioretinal neuroprotection by augmenting the chaperone buffering capacity and the decline of ubiquitylated substrates against proteostatic impairments. Further, CY's moonlighting activity holds pan -therapeutic potential against neurodegeneration.

Microglial activation in an amyotrophic lateral sclerosis-like model caused by Ranbp2 loss and nucleocytoplasmic transport impairment in retinal ganglion neurons.

Nucleocytoplasmic transport is dysregulated in sporadic and familial amyotrophic lateral sclerosis (ALS) and retinal ganglion neurons (RGNs) are purportedly involved in ALS. The Ran-binding protein 2 (Ranbp2) controls rate-limiting steps of nucleocytoplasmic transport. Mice with Ranbp2 loss in Thy1+-motoneurons develop cardinal ALS-like motor traits, but the impairments in RGNs and the degree of dysfunctional consonance between RGNs and motoneurons caused by Ranbp2 loss are unknown. This will help to understand the role of nucleocytoplasmic transport in the differential vulnerability of neuronal cell types to ALS and to uncover non-motor endophenotypes with pathognomonic signs of ALS. Here, we ascertain Ranbp2's function and endophenotypes in RGNs of an ALS-like mouse model lacking Ranbp2 in motoneurons and RGNs. Thy1+-RGNs lacking Ranbp2 shared with motoneurons the dysregulation of nucleocytoplasmic transport. RGN abnormalities were comprised morphologically by soma hypertrophy and optic nerve axonopathy and physiologically by a delay of the visual pathway's evoked potentials. Whole-transcriptome analysis showed restricted transcriptional changes in optic nerves that were distinct from those found in sciatic nerves. Specifically, the level and nucleocytoplasmic partition of the anti-apoptotic and novel substrate of Ranbp2, Pttg1/securin, were dysregulated. Further, acetyl-CoA carboxylase 1, which modulates de novo synthesis of fatty acids and T-cell immunity, showed the highest up-regulation (35-fold). This effect was reflected by the activation of ramified CD11b+ and CD45+-microglia, increase of F4\80+-microglia and a shift from pseudopodial/lamellipodial to amoeboidal F4\80+-microglia intermingled between RGNs of naive mice. Further, there was the intracellular sequestration in RGNs of metalloproteinase-28, which regulates macrophage recruitment and polarization in inflammation. Hence, Ranbp2 genetic insults in RGNs and motoneurons trigger distinct paracrine signaling likely by the dysregulation of nucleocytoplasmic transport of neuronal-type selective substrates. Immune-modulators underpinning RGN-to-microglial signaling are regulated by Ranbp2, and this neuronal-glial system manifests endophenotypes that are likely useful in the prognosis and diagnosis of motoneuron diseases, such as ALS.

Impairments in age-dependent ubiquitin proteostasis and structural integrity of selective neurons by uncoupling Ran GTPase from the Ran-binding domain 3 of Ranbp2 and identification of novel mitochondrial isoforms of ubiquitin-conjugating enzyme E2I (ubc9) and Ranbp2.

The Ran-binding protein 2 (Ranbp2/Nup358) is a cytoplasmic and peripheral nucleoporin comprised of 4 Ran-GTP-binding domains (RBDs) that are interspersed among diverse structural domains with multifunctional activities. Our prior studies found that the RBD2 and RBD3 of Ranbp2 control mitochondrial motility independently of Ran-GTP-binding in cultured cells, whereas loss of Ran-GTP-binding to RBD2 and RBD3 are essential to support cone photoreceptor development and the survival of mature retinal pigment epithelium (RPE) in mice. Here, we uncover that loss of Ran-GTP-binding to RBD3 alone promotes the robust age-dependent increase of ubiquitylated substrates and S1 subunit (Pmsd1) of the 19S cap of the proteasome in the retina and RPE and that such loss in RBD3 also compromises the structural integrity of the outer segment compartment of cone photoreceptors only and without affecting the viability of these neurons. We also found that the E2-ligase and partner of Ranbp2, ubc9, is localized prominently in the mitochondrial-rich ellipsoid compartment of photoreceptors, where Ranbp2 is also known to localize with and modulate the activity of mitochondrial proteins. However, the natures of Ranbp2 and ubc9 isoforms to the mitochondria are heretofore elusive. Subcellular fractionation, co-immunolocalization and immunoaffinity purification of Ranbp2 complexes show that novel isoforms of Ranbp2 and ubc9 with molecular masses distinct from the large Ranbp2 and unmodified ubc9 isoforms localize specifically to the mitochondrial fraction or associate with mitochondrial components, whereas unmodified and SUMOylated Ran GTPase are excluded from the mitochondrial fraction. Further, liposome-mediated intracellular delivery of an antibody against a domain shared by the mitochondrial and nuclear pore isoforms of Ranbp2 causes the profound fragmentation of mitochondria and their delocalization from Ranbp2 and without affecting Ranbp2 localization at the nuclear pores. Collectively, the data support that Ran GTPase-dependent and independent and moonlighting roles of Ranbp2 or domains thereof and ubc9 control selectively age-dependent, neural-type and mitochondrial functions.

Loss of Ranbp2 in motoneurons causes disruption of nucleocytoplasmic and chemokine signaling, proteostasis of hnRNPH3 and Mmp28, and development of amyotrophic lateral sclerosis-like syndromes.

The pathogenic drivers of sporadic and familial motor neuron disease (MND), such amyotrophic lateral sclerosis (ALS), are unknown. MND impairs the Ran GTPase cycle, which controls nucleocytoplasmic transport, ribostasis and proteostasis; however, cause-effect mechanisms of Ran GTPase modulators in motoneuron pathobiology have remained elusive. The cytosolic and peripheral nucleoporin Ranbp2 is a crucial regulator of the Ran GTPase cycle and of the proteostasis of neurological disease-prone substrates, but the roles of Ranbp2 in motoneuron biology and disease remain unknown. This study shows that conditional ablation of Ranbp2 in mouse Thy1 motoneurons causes ALS syndromes with hypoactivity followed by hindlimb paralysis, respiratory distress and, ultimately, death. These phenotypes are accompanied by: a decline in the nerve conduction velocity, free fatty acids and phophatidylcholine of the sciatic nerve; a reduction in the g-ratios of sciatic and phrenic nerves; and hypertrophy of motoneurons. Furthermore, Ranbp2 loss disrupts the nucleocytoplasmic partitioning of the import and export nuclear receptors importin ß and exportin 1, respectively, Ran GTPase and histone deacetylase 4. Whole-transcriptome, proteomic and cellular analyses uncovered that the chemokine receptor Cxcr4, its antagonizing ligands Cxcl12 and Cxcl14, and effector, latent and activated Stat3 all undergo early autocrine and proteostatic deregulation, and intracellular sequestration and aggregation as a result of Ranbp2 loss in motoneurons. These effects were accompanied by paracrine and autocrine neuroglial deregulation of hnRNPH3 proteostasis in sciatic nerve and motoneurons, respectively, and post-transcriptional downregulation of metalloproteinase 28 in the sciatic nerve. Mechanistically, our results demonstrate that Ranbp2 controls nucleocytoplasmic, chemokine and metalloproteinase 28 signaling, and proteostasis of substrates that are crucial to motoneuronal homeostasis and whose impairments by loss of Ranbp2 drive ALS-like syndromes.

Uncoupling phototoxicity-elicited neural dysmorphology and death by insidious function and selective impairment of Ran-binding protein 2 (Ranbp2).

Morphological disintegration of neurons is coupled invariably to neural death. In particular, disruption of outer segments of photoreceptor neurons triggers photoreceptor death regardless of the pathological stressors. We show that Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) mice with mutations in SUMO-binding motif (SBM) of cyclophilin-like domain (CLD) of Ran-binding protein 2 (Ranbp2) expressed in a null Ranbp2 background lack untoward effects in photoreceptors in the absence of light-stress. However, compared to wild type photoreceptors, light-stress elicits profound disintegration of outer segments of Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) with paradoxical age-dependent resistance of photoreceptors to death and genotype-independent activation of caspases. Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) exhibit photoreceptor death-independent changes in ubiquitin-proteasome system (UPS), but death-dependent increase of ubiquitin carrier protein 9(ubc9) levels. Hence, insidious functional impairment of SBM of Ranbp2's CLD promotes neuroprotection and uncoupling of photoreceptor degeneration and death against phototoxicity.

Targeting the cyclophilin domain of Ran-binding protein 2 (Ranbp2) with novel small molecules to control the proteostasis of STAT3, hnRNPA2B1 and M-opsin.

Cyclophilins are peptidyl cis-trans prolyl isomerases (PPIases), whose activity is typically inhibited by cyclosporine A (CsA), a potent immunosuppressor. Cyclophilins are also chaperones. Emerging evidence supports that cyclophilins present nonoverlapping PPIase and chaperone activities. The proteostasis of the disease-relevant substrates, signal transducer and activator of transcription 3 and 5 (STAT3/STAT5), heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), and M-opsin, is regulated by nonoverlapping chaperone and PPIase activities of the cyclophilin domain (CY) of Ranbp2, a multifunctional and modular scaffold that controls nucleocytoplasmic shuttling and proteostasis of selective substrates. Although highly homologous, CY and the archetypal cyclophilin A (CyPA) present distinct catalytic and CsA-binding activities owing to unique structural features between these cylophilins. We explored structural idiosyncrasies between CY and CyPA to screen in silico nearly 9 million small molecules (SM) against the CY PPIase pocket and identify SMs with selective bioactivity toward STAT3, hnRNPA2B1, or M-opsin proteostasis. We found three classes of SMs that enhance the cytokine-stimulated transcriptional activity of STAT3 without changing latent and activated STAT3 levels, down-regulate hnRNPA2B1 or M-opsin proteostasis, or a combination of these. Further, a SM that suppresses hnRNPA2B1 proteostasis also inhibits strongly and selectively the PPIase activity of CY. This study unravels chemical probes for multimodal regulation of CY of Ranbp2 and its substrates, and this regulation likely results in the allosterism stemming from the interconversion of conformational substates of cyclophilins. The results also demonstrate the feasibility of CY in drug discovery against disease-relevant substrates controlled by Ranbp2, and they open new opportunities for therapeutic interventions.

Selective impairment of a subset of Ran-GTP-binding domains of ran-binding protein 2 (Ranbp2) suffices to recapitulate the degeneration of the retinal pigment epithelium (RPE) triggered by Ranbp2 ablation.

Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2(-/-), with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2(-/-) background. Among the transgenic lines produced, only Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-)-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2(-/-). By contrast, Tg(RBD2/3*-HA) expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2(-/-) and Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-) share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting RPE degeneration.

Differential loss of prolyl isomerase or chaperone activity of Ran-binding protein 2 (Ranbp2) unveils distinct physiological roles of its cyclophilin domain in proteostasis.

The immunophilins, cyclophilins, catalyze peptidyl cis-trans prolyl-isomerization (PPIase), a rate-limiting step in protein folding and a conformational switch in protein function. Cyclophilins are also chaperones. Noncatalytic mutations affecting the only cyclophilins with known but distinct physiological substrates, the Drosophila NinaA and its mammalian homolog, cyclophilin-B, impair opsin biogenesis and cause osteogenesis imperfecta, respectively. However, the physiological roles and substrates of most cyclophilins remain unknown. It is also unclear if PPIase and chaperone activities reflect distinct cyclophilin properties. To elucidate the physiological idiosyncrasy stemming from potential cyclophilin functions, we generated mice lacking endogenous Ran-binding protein-2 (Ranbp2) and expressing bacterial artificial chromosomes of Ranbp2 with impaired C-terminal chaperone and with (Tg-Ranbp2(WT-HA)) or without PPIase activities (Tg-Ranbp2(R2944A-HA)). The transgenic lines exhibit unique effects in proteostasis. Either line presents selective deficits in M-opsin biogenesis with its accumulation and aggregation in cone photoreceptors but without proteostatic impairment of two novel Ranbp2 cyclophilin partners, the cytokine-responsive effectors, STAT3/STAT5. Stress-induced STAT3 activation is also unaffected in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). Conversely, proteomic analyses found that the multisystem proteinopathy/amyotrophic lateral sclerosis proteins, heterogeneous nuclear ribonucleoproteins A2/B1, are down-regulated post-transcriptionally only in Tg-Ranbp2(R2944A-HA)::Ranbp2(-/-). This is accompanied by the age- and tissue-dependent reductions of diubiquitin and ubiquitylated proteins, increased deubiquitylation activity, and accumulation of the 26 S proteasome subunits S1 and S5b. These manifestations are absent in another line, Tg-Ranbp2(CLDm-HA)::Ranbp2(-/-), harboring SUMO-1 and S1-binding mutations in the Ranbp2 cyclophilin-like domain. These results unveil distinct mechanistic and biological links between PPIase and chaperone activities of Ranbp2 cyclophilin toward proteostasis of selective substrates and with novel therapeutic potential.

Distinct and atypical intrinsic and extrinsic cell death pathways between photoreceptor cell types upon specific ablation of Ranbp2 in cone photoreceptors.

Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+ -apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2.

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms.

Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR) through a domain homologous to RCC1 (RHD), a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID) of RPGRIP1 embraces multivalently the shared RHD of RPGR(1-19) and RPGR(ORF15) isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α(1) expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α(1) self-aggregation ensue. RPGR(1-19) localizes to the endoplasmic reticulum, whereas RPGR(ORF15) presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α(1). Disease mutations in RPGR(1) (-19), RPGR(ORF15), or RID of RPGRIP1α(1), singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α(1) with RPGR(1-19) or RPGR(ORF15) in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGR(ORF15), but not RPGR(1-19), protects the RID of RPGRIP1α(1) from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α(1,) with deficits in RPGR(ORF15)-dependent intracellular localization of RPGRIP1α(1) contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies.

Ranbp2 haploinsufficiency mediates distinct cellular and biochemical phenotypes in brain and retinal dopaminergic and glia cells elicited by the Parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Many components and pathways transducing multifaceted and deleterious effects of stress stimuli remain ill-defined. The Ran-binding protein 2 (RanBP2) interactome modulates the expression of a range of clinical and cell-context-dependent manifestations upon a variety of stressors. We examined the role of Ranbp2 haploinsufficiency on cellular and metabolic manifestations linked to tyrosine-hydroxylase (TH(+)) dopaminergic neurons and glial cells of the brain and retina upon acute challenge to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonian neurotoxin, which models facets of Parkinson disease. MPTP led to stronger akinetic parkinsonism and slower recovery in Ranbp2 (+/-) than wild-type mice without viability changes of brain TH(+)-neurons of either genotype, with the exception of transient nuclear atypia via changes in chromatin condensation of Ranbp2 (+/-) TH(+)-neurons. Conversely, the number of wild-type retinal TH(+)-amacrine neurons compared to Ranbp2 (+/-) underwent milder declines without apoptosis followed by stronger recoveries without neurogenesis. These phenotypes were accompanied by a stronger rise of EdU(+)-proliferative cells and non-proliferative gliosis of GFAP(+)-Müller cells in wild-type than Ranbp2 (+/-) that outlasted the MPTP-insult. Finally, MPTP-treated wild-type and Ranbp2 (+/-) mice present distinct metabolic footprints in the brain or selective regions thereof, such as striatum, that are supportive of RanBP2-mediated regulation of interdependent metabolic pathways of lysine, cholesterol, free-fatty acids, or their ß-oxidation. These studies demonstrate contrasting gene-environment phenodeviances and roles of Ranbp2 between dopaminergic and glial cells of the brain and retina upon oxidative stress-elicited signaling and factors triggering a continuum of metabolic and cellular manifestations and proxies linked to oxidative stress, and chorioretinal and neurological disorders such as Parkinson.

Neuroprotection resulting from insufficiency of RANBP2 is associated with the modulation of protein and lipid homeostasis of functionally diverse but linked pathways in response to oxidative stress.

Oxidative stress is a deleterious stressor associated with a plethora of disease and aging manifestations, including neurodegenerative disorders, yet very few factors and mechanisms promoting the neuroprotection of photoreceptor and other neurons against oxidative stress are known. Insufficiency of RAN-binding protein-2 (RANBP2), a large, mosaic protein with pleiotropic functions, suppresses apoptosis of photoreceptor neurons upon aging and light-elicited oxidative stress, and promotes age-dependent tumorigenesis by mechanisms that are not well understood. Here we show that, by downregulating selective partners of RANBP2, such as RAN GTPase, UBC9 and ErbB-2 (HER2; Neu), and blunting the upregulation of a set of orphan nuclear receptors and the light-dependent accumulation of ubiquitylated substrates, light-elicited oxidative stress and Ranbp2 haploinsufficiency have a selective effect on protein homeostasis in the retina. Among the nuclear orphan receptors affected by insufficiency of RANBP2, we identified an isoform of COUP-TFI (Nr2f1) as the only receptor stably co-associating in vivo with RANBP2 and distinct isoforms of UBC9. Strikingly, most changes in proteostasis caused by insufficiency of RANBP2 in the retina are not observed in the supporting tissue, the retinal pigment epithelium (RPE). Instead, insufficiency of RANBP2 in the RPE prominently suppresses the light-dependent accumulation of lipophilic deposits, and it has divergent effects on the accumulation of free cholesterol and free fatty acids despite the genotype-independent increase of light-elicited oxidative stress in this tissue. Thus, the data indicate that insufficiency of RANBP2 results in the cell-type-dependent downregulation of protein and lipid homeostasis, acting on functionally interconnected pathways in response to oxidative stress. These results provide a rationale for the neuroprotection from light damage of photosensory neurons by RANBP2 insufficiency and for the identification of novel therapeutic targets and approaches promoting neuroprotection.

Gene and protein expression pilot profiling and biomarkers in an experimental mouse model of hypertensive glaucoma.

Glaucoma is a group of genetically heterogeneous neurodegenerative disorders causing the degeneration of the ganglion neurons of the retina. Increased intraocular pressure (IOP) is a hallmark risk factor promoting the death of ganglion neurons of the retina in glaucoma. Yet, the molecular processes underlying the degeneration of these neurons by increased IOP are not understood. To gain insight into the early molecular events and discover biomarkers induced by IOP, we performed gene and protein expression profiling to compare retinas of eyes with and without high IOP in a rodent model of experimental glaucoma. This pilot study found that the IOP-mediated changes in the transcription levels of a restricted set of genes implicated in peroxisomal and mitochondrial function, modulation of neuron survival and inflammatory processes, were also accompanied by changes in the levels of proteins encoded by the same genes. With the exception of the inflammatory markers, serum amyloid-A1 (SAA1) and serum amyloid-A2 (SAA2), the IOP-induced changes in protein expression were restricted to ganglion neurons of the retina and they were detected also in the vitreous, thus suggesting an early IOP-mediated loss of ganglion cell integrity. Interestingly, SAA1 and SAA2 were induced in retinal microglia cells, whereas they were reduced in sera of IOP-responsive mice. Hence, this study defines novel IOP-induced molecular processes, biomarkers and sources thereof, and it further validates the extension of the analyses herein reported to other genes modulated by IOP.

RANBP2 is an allosteric activator of the conventional kinesin-1 motor protein, KIF5B, in a minimal cell-free system.

The association of cargoes to kinesins is thought to promote kinesin activation, yet the validation of such a model with native cargoes is lacking because none is known to activate kinesins directly in an in vitro system of purified components. The RAN-binding protein 2 (RANBP2), through its kinesin-binding domain (KBD), associates in vivo with kinesin-1, KIF5B/KIF5C. Here, we show that KBD and its flanking domains, RAN GTPase-binding domains 2 and 3 (RBD2/RBD3), activate the ATPase activity of KIF5B approximately 30-fold in the presence of microtubules and ATP. The activation kinetics of KIF5B by RANBP2 is biphasic and highly cooperative. Deletion of one of its RBDs lowers the activation of KIF5B threefold and abolishes cooperativity. Remarkably, RBD2-KBD-RBD3 induces unfolding and modest activation of KIF5B in the absence of microtubules. Hence, RANBP2 is the first native and positive allosteric activator known to jump-start and boost directly the activity of a kinesin.

Association of the kinesin-binding domain of RanBP2 to KIF5B and KIF5C determines mitochondria localization and function.

The Ran-binding protein 2 (RanBP2) is a large mosaic protein with a pleiotropic role in cell function. Although the contribution of each partner and domain of RanBP2 to its biological functions are not understood, physiological deficits of RanBP2 downregulate glucose catabolism and energy homeostasis and lead to delocalization of mitochondria components in photosensory neurons. The kinesin-binding domain (KBD) of RanBP2 associates selectively in the central nervous system (CNS), and directly, with the ubiquitous and CNS-specific kinesins, KIF5B and KIF5C, respectively, but not with the highly homologous KIF5A. Here, we determine the molecular and biological bases of the selective interaction between RanBP2 and KIF5B/KIF5C. This interaction is conferred by a approximately 100-residue segment, comprising a portion of the coiled-coil and globular tail cargo-binding domains of KIF5B/KIF5C. A single residue conserved in KIF5B and KIF5C, but not KIF5A, confers KIF5-isotype-specific association with RanBP2. This interaction is also mediated by a conserved leucine-like heptad motif present in KIF5s and KBD of RanBP2. Selective inhibition of the interaction between KBD of RanBP2 and KIF5B/KIF5C in cell lines causes perinuclear clustering of mitochondria, but not of lysosomes, deficits in mitochondrial membrane potential and ultimately, cell shrinkage. Collectively, the data provide a rationale of the KIF5 subtype-specific interaction with RanBP2 and support a novel kinesin-dependent role of RanBP2 in mitochondria transport and function. The data also strengthen a model whereby the selection of a large array of cargoes for transport by a restricted number of motor proteins is mediated by adaptor proteins such as RanBP2.

Cochlear pathology of the circling mouse: a new mouse model of DFNB6.

CONCLUSION: The circling mouse (cir/cir) has phenotypes which follow the pattern of neuroepithelial defects of deafness from 10 days after birth. The cir mouse is defective in Tmie gene, the function of which should be further elucidated. OBJECTIVES: We previously reported a recessive mutation of deafness called circling mice (cir/cir). The present study focused on investigating phenotypes and histological findings of the cochlea in circling mice with respect to age. MATERIALS AND METHODS: In order to analyze cochlear pathology over time, five different age groups of circling mice were examined (10, 18, 21, 35, and 90 days old). The organs of Corti and spiral ganglion neurons in basal and middle turns were evaluated. RESULTS: The pathology of the organ of Corti followed the pattern of neuroepithelial defects. Hair cells in organs of Corti had degenerated in circling mice at 10 days old, in a time-dependent manner. Scanning electron microscopy (SEM) showed that stereociliary bundles were irregular in size and had shortened at 10 days, and that this degeneration was complete at 21 days. The number of spiral ganglion neurons significantly reduced with age. RT-PCR analysis indicated that the transmembrane inner ear gene (Tmie) was absent in various organs in circling mice.

The circling mouse (C57BL/6J-cir) has a 40-kilobase genomic deletion that includes the transmembrane inner ear (tmie) gene.

The circling mouse (C57BL6-cir) shows deafness and circling behavior in homozygotes. The mutation is transmitted with 100% penetrance by an autosomal recessive gene on chromosome 9. In the present study, we characterized the circling mutation as a 40-kilobase deletion that includes the transmembrane inner ear (tmie) gene. The tmie gene was first identified because its mutation causes deafness and circling behavior in spinner mice. We suggest that the genomic deletion of circling mice is a different, but allelic, mutation to that of spinner mice. In addition, during general behavioral investigations for complementation tests of the 2 strains, we found that circling and spinner mice may differ in their behavioral responses to a new environment.

Human keratin 14 driven HPV 16 E6/E7 transgenic mice exhibit hyperkeratinosis.

Human papillomavirus type 16 (HPV16) has been known as a major causative factor for the development of uterine cervical carcinomas. To investigate the in vivo activity of HPV16 expressed in squamous epithelia, transgenic mice harboring HPV16 E6/E7 with human keratin 14 (hK14) promoter were generated. Grossly, hK14 driven HPV16 E6/E7 transgenic mice exhibited multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair in neonates, thickened ears, and loss of hair in adults. Transgenic mice with phenotype exhibiting severe wrinkled skin and a lack of hair growth died at the age of 3-4 weeks. Histological analysis revealed that in transgenic mice survived beyond the initial 3-4 weeks, HPV16 E6/E7 causes epidermal hyperplasia in multiple transgenic lineages with high incidence of transgene penetration. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and keratinocytes, and was associated with hyperkeratosis. Such activities were significantly higher in the skin of transgenic mice than that of the normal mice. Thus, these transgenic mice appeared to be useful for the expression of HPV16 E6/E7 gene and subsequent analysis on hyperkeratosis.