Shaping the brain vasculature in development and disease in the single-cell era.

The CNS critically relies on the formation and proper function of its vasculature during development, adult homeostasis and disease. Angiogenesis - the formation of new blood vessels - is highly active during brain development, enters almost complete quiescence in the healthy adult brain and is reactivated in vascular-dependent brain pathologies such as brain vascular malformations and brain tumours. Despite major advances in the understanding of the cellular and molecular mechanisms driving angiogenesis in peripheral tissues, developmental signalling pathways orchestrating angiogenic processes in the healthy and the diseased CNS remain incompletely understood. Molecular signalling pathways of the 'neurovascular link' defining common mechanisms of nerve and vessel wiring have emerged as crucial regulators of peripheral vascular growth, but their relevance for angiogenesis in brain development and disease remains largely unexplored. Here we review the current knowledge of general and CNS-specific mechanisms of angiogenesis during brain development and in brain vascular malformations and brain tumours, including how key molecular signalling pathways are reactivated in vascular-dependent diseases. We also discuss how these topics can be studied in the single-cell multi-omics era.

Circulating small extracellular vesicles mediate vascular hyperpermeability in diabetes.

AIMS/HYPOTHESIS: A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier. METHODS: sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors. RESULTS: Using intravital imaging, we found that diabetic mice (Leprdb/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell-cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state. CONCLUSIONS/INTERPRETATION: Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.

Parkinson's disease pathology is directly correlated to SIRT3 in human subjects and animal models: Implications for AAV.SIRT3-myc as a disease-modifying therapy.

In Parkinson's disease (PD), post-mortem studies in affected brain regions have demonstrated a decline in mitochondrial number and function. This combined with many studies in cell and animal models suggest that mitochondrial dysfunction is central to PD pathology. We and others have shown that the mitochondrial protein deacetylase, SIRT3, has neurorestorative effects in PD models. In this study, to determine whether there is a link between PD pathology and SIRT3, we analysed SIRT3 levels in human subjects with PD, and compared to age-matched controls. In the SNc of PD subjects, SIRT3 was reduced by 56.8 ± 15.5% compared to control, regardless of age (p < 0.05, R = 0.6539). Given that age is the primary risk factor for PD, this finding suggests that reduced SIRT3 may contribute to PD pathology. Next, we measured whether there was a correlation between α-synuclein and SIRT3. In a parallel study, we assessed the disease-modifying potential of SIRT3 over-expression in a seeding model of α-synuclein. In PFF rats, infusion of rAAV1.SIRT3-myc reduced abundance of α-synuclein inclusions by 30.1 ± 18.5%. This was not observed when deacetylation deficient SIRT3H248Y was transduced, demonstrating the importance of SIRT3 deacetylation in reducing α-synuclein aggregation. These studies confirm that there is a clear difference in SIRT3 levels in subjects with PD compared to age-matched controls, suggesting a link between SIRT3 and the progression of PD. We also demonstrate that over-expression of SIRT3 reduces α-synuclein aggregation, further validating AAV.SIRT3-myc as a potential disease-modifying solution for PD.

Delayed administration of elezanumab, a human anti-RGMa neutralizing monoclonal antibody, promotes recovery following cervical spinal cord injury.

Spinal cord injury (SCI) elicits a cascade of degenerative events including cell death, axonal degeneration, and the upregulation of inhibitory molecules which limit repair. Repulsive guidance molecule A (RGMa) is an axon growth inhibitor which is also involved in neuronal cell death and differentiation. SCI causes upregulation of RGMa in the injured rodent, non-human primate, and human spinal cord. Recently, we showed that delayed administration of elezanumab, a high affinity human RGMa-specific monoclonal antibody, promoted neuroprotective and regenerative effects following thoracic SCI. Since most human traumatic SCI is at the cervical level, and level-dependent anatomical and molecular differences may influence pathophysiological responses to injury and treatment, we examined the efficacy of elezanumab and its therapeutic time window of administration in a clinically relevant rat model of cervical impact-compression SCI. Pharmacokinetic analysis of plasma and spinal cord tissue lysate showed comparable levels of RGMa antibodies with delayed administration following cervical SCI. At 12w after SCI, elezanumab promoted long term benefits including perilesional sparing of motoneurons and increased neuroplasticity of key descending pathways involved in locomotion and fine motor function. Elezanumab also promoted growth of corticospinal axons into spinal cord gray matter and enhanced serotonergic innervation of the ventral horn to form synaptic connections caudal to the cervical lesion. Significant recovery in grip and trunk/core strength, locomotion and gait, and spontaneous voiding ability was found in rats treated with elezanumab either immediately post-injury or at 3 h post-SCI, and improvements in specific gait parameters were found when elezanumab was delayed to 24 h post-injury. We also developed a new locomotor score, the Cervical Locomotor Score, a simple and sensitive measure of trunk/core and limb strength and stability during dynamic locomotion.

Cholesterol synthesis inhibition promotes axonal regeneration in the injured central nervous system.

Neuronal regeneration in the injured central nervous system is hampered by multiple extracellular proteins. These proteins exert their inhibitory action through interactions with receptors that are located in cholesterol rich compartments of the membrane termed lipid rafts. Here we show that cholesterol-synthesis inhibition prevents the association of the Neogenin receptor with lipid rafts. Furthermore, we show that cholesterol-synthesis inhibition enhances axonal growth both on inhibitory -myelin and -RGMa substrates. Following optic nerve injury, lowering cholesterol synthesis with both drugs and siRNA-strategies allows for robust axonal regeneration and promotes neuronal survival. Cholesterol inhibition also enhanced photoreceptor survival in a model of Retinitis Pigmentosa. Our data reveal that Lovastatin leads to several opposing effects on regenerating axons: cholesterol synthesis inhibition promotes regeneration whereas altered prenylation impairs regeneration. We also show that the lactone prodrug form of lovastatin has differing effects on regeneration when compared to the ring-open hydroxy-acid form. Thus the association of cell surface receptors with lipid rafts contributes to axonal regeneration inhibition, and blocking cholesterol synthesis provides a potential therapeutic approach to promote neuronal regeneration and survival in the diseased Central Nervous System. SIGNIFICANCE STATEMENT: Statins have been intensively used to treat high levels of cholesterol in humans. However, the effect of cholesterol inhibition in both the healthy and the diseased brain remains controversial. In particular, it is unclear whether cholesterol inhibition with statins can promote regeneration and survival following injuries. Here we show that late stage cholesterol inhibition promotes robust axonal regeneration following optic nerve injury. We identified distinct mechanisms of action for activated vs non-activated Lovastatin that may account for discrepancies found in the literature. We show that late stage cholesterol synthesis inhibition alters Neogenin association with lipid rafts, thereby i) neutralizing the inhibitory function of its ligand and ii) offering a novel opportunity to promote CNS regeneration and survival following injuries.

Extracellular phosphorylation drives the formation of neuronal circuitry.

Until recently, the existence of extracellular kinase activity was questioned. Many proteins of the central nervous system are targeted, but it remains unknown whether, or how, extracellular phosphorylation influences brain development. Here we show that the tyrosine kinase vertebrate lonesome kinase (VLK), which is secreted by projecting retinal ganglion cells, phosphorylates the extracellular protein repulsive guidance molecule b (RGMb) in a dorsal-ventral descending gradient. Silencing of VLK or RGMb causes aberrant axonal branching and severe axon misguidance in the chick optic tectum. Mice harboring RGMb with a point mutation in the phosphorylation site also display aberrant axonal pathfinding. Mechanistic analyses show that VLK-mediated RGMb phosphorylation modulates Wnt3a activity by regulating LRP5 protein gradients. Thus, the secretion of VLK by projecting neurons provides crucial signals for the accurate formation of nervous system circuitry. The dramatic effect of VLK on RGMb and Wnt3a signaling implies that extracellular phosphorylation likely has broad and profound effects on brain development, function and disease.

Delayed administration of the human anti-RGMa monoclonal antibody elezanumab promotes functional recovery including spontaneous voiding after spinal cord injury in rats.

Spinal cord injury (SCI) often results in permanent functional loss due to a series of degenerative events including cell death, axonal damage, and the upregulation of inhibitory proteins that impede regeneration. Repulsive Guidance Molecule A (RGMa) is a potent inhibitor of axonal growth that is rapidly upregulated following injury in both the rodent and human central nervous system (CNS). Previously, we showed that monoclonal antibodies that specifically block inhibitory RGMa signaling promote neuroprotective and regenerative effects when administered acutely in a clinically relevant rat model of thoracic SCI. However, it is unknown whether systemic administration of RGMa blocking antibodies are effective for SCI after delayed administration. Here, we administered elezanumab, a human monoclonal antibody targeting RGMa, intravenously either acutely or at 3 h or 24 h following thoracic clip impact-compression SCI. Rats treated with elezanumab acutely and at 3 h post-injury showed improvements in overground locomotion and fine motor function and gait. Rats treated 24 h post-SCI trended towards better recovery demonstrating significantly greater stride length and swing speed. Treated rats also showed greater tissue preservation with reduced lesion areas. As seen with acute treatment, delayed administration of elezanumab at 3 h post-SCI also increased perilesional neuronal sparing and serotonergic and corticospinal axonal plasticity. In addition, all elezanumab treated rats showed earlier spontaneous voiding ability and less post-trauma bladder wall hypertrophy. Together, our data demonstrate the therapeutic efficacy of delayed systemic administration of elezanumab in a rat model of SCI, and uncovers a new role for RGMa inhibition in bladder recovery following SCI.

Emerging evidence for cell-autonomous axon guidance.

Current models of axon guidance within the central nervous system (CNS) involve the presentation of environmental cues to navigating growth cones. The surrounding and target tissues present a variety of ligands that either restrict or promote growth, thus providing pathfinding instructions to developing axons. Recent findings show that RGMb, a GPI anchored extracellular protein present on retinal ganglion cells, down-regulates Wnt3a signaling by lowering LRP5 levels at the membrane surface. When RGMb is phosphorylated by the extracellular tyrosine kinase VLK, phosphorylated RGMb (p-RGMb) is internalized and carries LRP5 towards intracellular compartments. In the eye, a dorsal-high ventral-low gradient of VLK generates a dorsal-low ventral-high gradient of LRP5 that modulates Wnt3a signaling. These molecules, which are all expressed by individual RGCs, generate Wnt-signal gradients along the dorso-ventral axis of the retina, resulting in differential axon growth which in turn regulates proper retino-tectal/collicular map formation. This pathway represents a regulatory mechanism whereby extracellular phosphorylation generates what may be the first example of a unique self-guiding mechanism that affects neuronal-target connections independent of paracrine signals from the surrounding target tissue.

C2 Domains of Munc13-4 Are Crucial for Ca2+-Dependent Degranulation and Cytotoxicity in NK Cells.

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.

UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans.

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

Neurosurgical Modeling of Retinal Ischemia-Reperfusion Injury.

BACKGROUND: A reliable model of ischemia-reperfusion is required to evaluate the efficacy and safety of neuroprotective therapies for stroke. We present a novel reproducible pterygopalatine-ophthalmic artery ligation model of ischemia-reperfusion injury in the retina. METHODS: Rats were subjected to ophthalmic artery/meningeal sheath ligation (OAML-standard method) or clamping of the pterygopalatine-ophthalmic artery (OAC-new method) for 30 minutes. Retinal ganglion cell (RGC) survival was assessed by prelabeling with FluoroGold (FG) (Santa Cruz Biotechnology, CA, USA) and RNA-binding protein with multiple splicing (RBPMS) at 14 days after ischemia, and all results were compared with a sham group (n = 7 in each group). RESULTS: RGC density in the normal-uninjured (FG-labeled) group was 2111 ± 38 cells/mm2 (mean ± standard error of mean) and that in the RBPMS-labeled group was 2142 ± 35 cells/mm2. The OAML procedure significantly reduced RGC density to 738 ± 23 cells/mm2 and 780 ± 41 cells/mm2 (P < .001) in the FG-labeled and RBPMS-labeled groups, respectively. Similarly, OAC reduced RGC survival to 782 ± 19 cells/mm2 and 813 ± 22 cells/mm2 (P < .001) in the FG-labeled and RBPMS-labeled groups, respectively. RGC survival was similar following OAC and OAML models, suggesting that both induce comparable levels of damage. However, RGC survival in the OAC model was found to have less dispersion than OAML-induced ischemia. CONCLUSIONS: These results suggest that the OAC procedure is a reliable reproduction of ischemia-reperfusion injury that mimics the effects of ophthalmic artery occlusion in humans and provides a useful research model for testing manipulations directed against pathways involved in RGC ischemic degeneration.

Rescue of behavioral and EEG deficits in male and female Mecp2-deficient mice by delayed Mecp2 gene reactivation.

Mutations of the X-linked gene encoding methyl CpG binding protein type 2 (MECP2) are the predominant cause of Rett syndrome, a severe neurodevelopmental condition that affects primarily females. Previous studies have shown that major phenotypic deficits arising from MeCP2-deficiency may be reversible, as the delayed reactivation of the Mecp2 gene in Mecp2-deficient mice improved aspects of their Rett-like phenotype. While encouraging for prospective gene replacement treatments, it remains unclear whether additional Rett syndrome co-morbidities recapitulated in Mecp2-deficient mice will be similarly responsive to the delayed reintroduction of functional Mecp2. Here, we show that the delayed reactivation of Mecp2 in both male and female Mecp2-deficient mice rescues established deficits in motor and anxiety-like behavior, epileptiform activity, cortical and hippocampal electroencephalogram patterning and thermoregulation. These findings indicate that neural circuitry deficits arising from the deficiency in Mecp2 are not engrained, and provide further evidence that delayed restoration of Mecp2 function can improve a wide spectrum of the Rett-like deficits recapitulated by Mecp2-deficient mice.

SNAP-25, but not SNAP-23, is essential for photoreceptor development, survival, and function in mice.

SNARE-mediated vesicular transport is thought to play roles in photoreceptor glutamate exocytosis and photopigment delivery. However, the functions of Synaptosomal-associated protein (SNAP) isoforms in photoreceptors are unknown. Here, we revisit the expression of SNAP-23 and SNAP-25 and generate photoreceptor-specific knockout mice to investigate their roles. Although we find that SNAP-23 shows weak mRNA expression in photoreceptors, SNAP-23 removal does not affect retinal morphology or vision. SNAP-25 mRNA is developmentally regulated and undergoes mRNA trafficking to photoreceptor inner segments at postnatal day 9 (P9). SNAP-25 knockout photoreceptors develop normally until P9 but degenerate by P14 resulting in severe retinal thinning. Photoreceptor loss in SNAP-25 knockout mice is associated with abolished electroretinograms and vision loss. We find mistrafficked photopigments, enlarged synaptic vesicles, and abnormal synaptic ribbons which potentially underlie photoreceptor degeneration. Our results conclude that SNAP-25, but not SNAP-23, mediates photopigment delivery and synaptic functioning required for photoreceptor development, survival, and function.

Spreading depolarization suppression from inter-astrocytic gap junction blockade assessed with multimodal imaging and a novel wavefront detection scheme.

Spreading depolarizations (SDs) are an enigmatic and ubiquitous co-morbidity of neural dysfunction. SDs are propagating waves of local field depolarization and increased extracellular potassium. They increase the metabolic demand on brain tissue, resulting in changes in tissue blood flow, and are associated with adverse neurological consequences including stroke, epilepsy, neurotrauma, and migraine. Their occurrence is associated with poor patient prognosis through mechanisms which are only partially understood. Here we show in vivo that two (structurally dissimilar) drugs, which suppress astroglial gap junctional communication, can acutely suppress SDs. We found that mefloquine hydrochloride (MQH), administered IP, slowed the propagation of the SD potassium waveform and intermittently led to its suppression. The hemodynamic response was similarly delayed and intermittently suppressed. Furthermore, in instances where SD led to transient tissue swelling, MQH reduced observable tissue displacement. Administration of meclofenamic acid (MFA) IP was found to reduce blood flow, both proximal and distal, to the site of SD induction, preceding a large reduction in the amplitude of the SD-associated potassium wave. We introduce a novel image processing scheme for SD wavefront localization under low-contrast imaging conditions permitting full-field wavefront velocity mapping and wavefront parametrization. We found that MQH administration delayed SD wavefront's optical correlates. These two clinically used drugs, both gap junctional blockers found to distinctly suppress SDs, may be of therapeutic benefit in the various brain disorders associated with recurrent SDs.

The liver and muscle secreted HFE2-protein maintains central nervous system blood vessel integrity.

Liver failure causes breakdown of the Blood CNS Barrier (BCB) leading to damages of the Central-Nervous-System (CNS), however the mechanisms whereby the liver influences BCB-integrity remain elusive. One possibility is that the liver secretes an as-yet to be identified molecule(s) that circulate in the serum to directly promote BCB-integrity. To study BCB-integrity, we developed light-sheet imaging for three-dimensional analysis. We show that liver- or muscle-specific knockout of Hfe2/Rgmc induces BCB-breakdown, leading to accumulation of toxic-blood-derived fibrinogen in the brain, lower cortical neuron numbers, and behavioral deficits in mice. Soluble HFE2 competes with its homologue RGMa for binding to Neogenin, thereby blocking RGMa-induced downregulation of PDGF-B and Claudin-5 in endothelial cells, triggering BCB-disruption. HFE2 administration in female mice with experimental autoimmune encephalomyelitis, a model for multiple sclerosis, prevented paralysis and immune cell infiltration by inhibiting RGMa-mediated BCB alteration. This study has implications for the pathogenesis and potential treatment of diseases associated with BCB-dysfunction.

The double-stranded RNA-binding protein Staufen 2 regulates eye size.

Regulation of tissue size is a poorly understood process. Mammalian Staufen 2 (Stau2) is a double-stranded mRNA binding protein known to regulate dendrite formation in vitro as well as cell survival and migration in vivo. Three Stau2 isoforms have been identified in the brain of mammals. Here we show that all these Stau2 isoforms are also expressed in the developing eye of chicken embryos. Strikingly, ectopic expression of Stau2 was sufficient to increase eye size, suggesting a novel biological role of Stau2 in eye morphogenesis. Moreover, down regulation of Stau2 in vivo resulted in a small eye. Microphthalmia was not associated with either increased cell death or differentiation but with reduced cell proliferation. Rescue experiments showed that all three Stau2 isoforms present in the developing eye could prevent microphthalmia. Finally, we showed that Stau2 silencing decreased HES-1 and Sox-2 in the developing eye. These data highlight a new biological function for Stau2 and suggest that translation control of specific Stau2-associated transcripts may be a key regulator of tissue size.

The Dyslexia-associated gene KIAA0319L is involved in neuronal migration in the developing chick visual system.

The gene KIAA0319-Like (KIAA0319L) is thought to confer susceptibility for developmental dyslexia. Dyslexia may be caused by alterations in neuronal migration, and in utero knockdown of KIAA0319L in rats indicated migration errors. However, studies carried out with KIAA0319L knockout mice did not reveal an altered neuronal migration phenotype. Gene knockout may activate compensatory mechanisms to buffer against genetic mutations during development. Here we assessed the role of KIAA0319L on migrating neurons in the chick developing tectum. Whole mount in situ hybridization was performed for KIAA0319L on embryonic day (E)3 - E5 chick embryos and in situ hybridization on sections was performed at later stages. The specificity and efficiency of engineered microRNA (miRNA) constructs targeting KIAA0319L for knocking down KIAA0319L were verified. miRNAs were electroporated into E5 chick optic tecta. Our studies demonstrate that KIAA0319L is expressed in the developing chick visual system, as well as in the otic vesicles. Knockdown of KIAA0319L in the optic tectum results in abnormal neuronal migration, strengthening the argument that KIAA0319L is involved in this developmental process.

Affinity-controlled release of rod-derived cone viability factor enhances cone photoreceptor survival.

Retinitis pigmentosa (RP) is a group of genetic diseases that results in rod photoreceptor cell degeneration, which subsequently leads to cone photoreceptor cell death, impaired vision and eventual blindness. Rod-derived cone viability factor (RdCVF) is a protein which has two isoforms: a short form (RdCVF) and a long form (RdCVFL) which act on cone photoreceptors in the retina. RdCVFL protects photoreceptors by reducing hyperoxia in the retina; however, sustained delivery of RdCVFL remains challenging. We developed an affinity-controlled release strategy for RdCVFL. An injectable physical blend of hyaluronan and methylcellulose (HAMC) was covalently modified with a peptide binding partner of the Src homology 3 (SH3) domain. This domain was expressed as a fusion protein with RdCVFL, thereby enabling its controlled release from HAMC-binding peptide. Sustained release of RdCVFL was demonstrated for the first time as RdCVFL-SH3 from HAMC-binding peptide for 7 d in vitro. To assess bioactivity, chick retinal dissociates were harvested and treated with the affinity-released recombinant protein from the HAMC-binding peptide vehicle. After 6 d in culture, cone cell viability was greater when cultured with released RdCVFL-SH3 relative to controls. We utilized computational fluid dynamics to model release of RdCVFL-SH3 from our delivery vehicle in the vitreous of the human eye. We demonstrate that our delivery vehicle can prolong the bioavailability of RdCVFL-SH3 in the retina, potentially enhancing its therapeutic effects. Our affinity-based system constitutes a versatile delivery platform for ultimate intraocular injection in the treatment of retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Retinitis pigmentosa (RP) is the leading cause of inherited blindness in the world. Rod-derived cone viability factor (RdCVF), a novel protein paracrine factor, is effective in preclinical models of RP. To extend its therapeutic effects, we developed an affinity-controlled release strategy for the long form of RdCVF, RdCVFL. We expressed RdCVFL as a fusion protein with an Src homology 3 domain (SH3). We then utilized a hydrogel composed of hyaluronan and methylcellulose (HAMC) and modified it with SH3 binding peptides to investigate its release in vitro. Furthermore, we designed a mathematical model of the human eye to investigate delivery of the protein from the delivery vehicle. This work paves the way for future investigation of controlled release RdCVF.

Double mutation of open syntaxin and UNC-18 P334A leads to excitatory-inhibitory imbalance and impairs multiple aspects of C. elegans behavior.

SNARE and Sec/Munc18 proteins are essential in synaptic vesicle exocytosis. Open form t-SNARE syntaxin and UNC-18 P334A are well-studied exocytosis-enhancing mutants. Here we investigate the interrelationship between the two mutations by generating double mutants in various genetic backgrounds in C. elegans. While each single mutation rescued the motility of CAPS/unc-31 and synaptotagmin/snt-1 mutants significantly, double mutations unexpectedly worsened motility or lost their rescuing effects. Electrophysiological analyses revealed that simultaneous mutations of open syntaxin and gain-of-function P334A UNC-18 induces a strong imbalance of excitatory over inhibitory transmission. In liposome fusion assays performed with mammalian proteins, the enhancement of fusion caused by the two mutations individually was abolished when the two mutations were introduced simultaneously, consistent with what we observed in C. elegans. We conclude that open syntaxin and P334A UNC-18 do not have additive beneficial effects, and this extends to C. elegans' characteristics such as motility, growth, offspring bared, body size, and exocytosis, as well as liposome fusion in vitro. Our results also reveal unexpected differences between the regulation of exocytosis in excitatory versus inhibitory synapses.

Deneddylation of ribosomal proteins promotes synergy between MLN4924 and chemotherapy to elicit complete therapeutic responses.

The neddylation inhibitor MLN4924/Pevonedistat is in clinical trials for multiple cancers. Efficacy is generally attributed to cullin RING ligase (CRL) inhibition, but the contribution of non-CRL targets is unknown. Here, CRISPR screens map MLN4924-monotherapy sensitivity in retinoblastoma to a classic DNA damage-induced p53/E2F3/BAX-dependent death effector network, which synergizes with Nutlin3a or Navitoclax. In monotherapy-resistant cells, MLN4924 plus standard-of-care topotecan overcomes resistance, but reduces DNA damage, instead harnessing ribosomal protein nucleolar-expulsion to engage an RPL11/p21/MYCN/E2F3/p53/BAX synergy network that exhibits extensive cross-regulation. Strikingly, unneddylatable RPL11 substitutes for MLN4924 to perturb nucleolar function and enhance topotecan efficacy. Orthotopic tumors exhibit complete responses while preserving visual function. Moreover, MLN4924 plus melphalan deploy this DNA damage-independent strategy to synergistically kill multiple myeloma cells. Thus, MLN4924 synergizes with standard-of-care drugs to unlock a nucleolar death effector network across cancer types implying broad therapeutic relevance.