Soluble Nogo-Receptor-Fc decoy (AXER-204) in patients with chronic cervical spinal cord injury in the USA: a first-in-human and randomised clinical trial.

BACKGROUND: Spinal cord injury (SCI) causes neural disconnection and persistent neurological deficits, so axon sprouting and plasticity might promote recovery. Soluble Nogo-Receptor-Fc decoy (AXER-204) blocks inhibitors of axon growth and promotes recovery of motor function after SCI in animals. This first-in-human and randomised trial sought to determine primarily the safety and pharmacokinetics of AXER-204 in individuals with chronic SCI, and secondarily its effect on recovery. METHODS: We conducted a two-part study in adults (aged 18-65 years) with chronic (>1 year) cervical traumatic SCI at six rehabilitation centres in the USA. In part 1, AXER-204 was delivered open label as single intrathecal doses of 3 mg, 30 mg, 90 mg, or 200 mg, with primary outcomes of safety and pharmacokinetics. Part 2 was a randomised, parallel, double-blind comparison of six intrathecal doses of 200 mg AXER-204 over 104 days versus placebo. Participants were randomly allocated (1:1) by investigators using a central electronic system, stratified in blocks of four by American Spinal Injury Association Impairment Scale grade and receipt of AXER-204 in part 1. All investigators and patients were masked to treatment allocation until at least day 169. The part 2 primary objectives were safety and pharmacokinetics, with a key secondary objective to assess change in International Standards for Neurological Classification of SCI (ISNCSCI) Upper Extremity Motor Score (UEMS) at day 169 for all enrolled participants. This trial is registered with ClinicalTrials.gov, NCT03989440, and is completed. FINDINGS: We treated 24 participants in part 1 (six per dose; 18 men, six women), and 27 participants in part 2 (13 placebo, 14 AXER-204; 23 men, four women), between June 20, 2019, and June 21, 2022. There were no deaths and no discontinuations from the study due to an adverse event in part 1 and 2. In part 2, treatment-related adverse events were of similar incidence in AXER-204 and placebo groups (ten [71%] vs nine [69%]). Headache was the most common treatment-related adverse event (five [21%] in part 1, 11 [41%] in part 2). In part 1, AXER-204 reached mean maximal CSF concentration 1 day after dosing with 200 mg of 412 000 ng/mL (SD 129 000), exceeding those concentrations that were efficacious in animal studies. In part 2, mean changes from baseline to day 169 in ISNCSCI UEMS were 1·5 (SD 3·3) for AXER-204 and 0·9 (2·3) for placebo (mean difference 0·54, 95% CI -1·48 to 2·55; p=0·59). INTERPRETATION: This study delivers the first, to our knowledge, clinical trial of a rationally designed pharmacological treatment intended to promote neural repair in chronic SCI. AXER-204 appeared safe and reached target CSF concentrations; exploratory biomarker results were consistent with target engagement and synaptic stabilisation. Post-hoc subgroup analyses suggest that future trials could investigate efficacy in patients with moderately severe SCI without prior AXER-204 exposure. FUNDING: Wings for Life Foundation, National Institute of Neurological Disorders and Stroke, National Center for Advancing Translational Sciences, National Institute on Drug Abuse, and ReNetX Bio.

Novel insights into the genetic profile of hereditary spastic paraplegia in India.

The Hereditary Spastic Paraplegias (HSPs) are a group of clinically and genetically heterogeneous disorders characterized by length dependent degeneration of the corticospinal tracts. Genetic data related to HSPs are limited from India. We aimed to comprehensively analyse the phenotypic characteristics and genetic basis of a large cohort of HSP from India. Patients with HSP phenotype were evaluated for their clinical features, electrophysiological and radiological abnormalities. Genetic analyses were carried out by clinical exome sequencing (n = 52) and targeted sequencing (n = 5). The cohort comprised of 57 probands (M:F 40:17, age: 3.5-49 years). Based on the phenotype, the cohort could be categorized as 'pure' (n = 15, 26.3%) and 'complicated' (n = 42, 73.7%) HSP. Brain MRI showed thin corpus callosum (n = 10), periventricular hyperintensities (n = 20), cerebral atrophy (n = 3), cerebellar atrophy (n = 3) and diffuse atrophy (n = 4). Sixty-seven variants representing 40 genes were identified including 47 novel variants. Forty-eight patients (84.2%) had variants in genes previously implicated in HSP and other spastic paraplegia syndromes (SPG genes = 24, non-SPG genes = 24); among these 13 had variations in more than one gene and 12 patients (21.0%) had variations in genes implicated in potentially treatable/modifiable metabolic disorders (MTHFR = 8, MTRR = 1, ARG1 = 2 and ABCD1 = 1). In nine patients, no genetic variants implicated in spastic paraplegia phenotype were identified. Thus, the present study from India highlights the phenotypic complexities and spectrum of genetic variations in patients with HSP including those implicated in metabolically modifiable disorders. It sets a platform for carrying out functional studies to validate the causal role of the novel variants and variants of uncertain significance.

Rabphilin3A reduces integrin-dependent growth cone signaling to restrict axon regeneration after trauma.

Neural repair after traumatic spinal cord injury depends upon the restoration of neural networks via axonal sprouting and regeneration. Our previous genome wide loss-of-function screen identified Rab GTPases as playing a prominent role in preventing successful axon sprouting and regeneration. Here, we searched for Rab27b interactors and identified Rabphilin3A as an effector within regenerating axons. Growth cone Rabphilin3a colocalized and physically associated with integrins at puncta in the proximal body of the axonal growth cone. In regenerating axons, loss of Rabphilin3a increased integrin enrichment in the growth cone periphery, enhanced focal adhesion kinase activation, increased F-actin-rich filopodial density and stimulated axon extension. Compared to wild type, mice lacking Rabphilin3a exhibited greater regeneration of retinal ganglion cell axons after optic nerve crush as well as greater corticospinal axon regeneration after complete thoracic spinal cord crush injury. After moderate spinal cord contusion injury, there was greater corticospinal regrowth in the absence of Rph3a. Thus, an endogenous Rab27b - Raphilin3a pathway limits integrin action in the growth cone, and deletion of this monomeric GTPase pathway permits reparative axon growth in the injured adult mammalian central nervous system.

Machine Learning Method Reveals Hidden Strong Metal-Support Interaction in Microscopy Datasets.

Forming an ultra-thin, permeable encapsulation oxide-support layer on a metal catalyst surface is considered an effective strategy for achieving a balance between high stability and high activity in heterogenous catalysts. The success of such a design relies not only on the thickness, ideally one to two atomic layers thick, but also on the morphology and chemistry of the encapsulation layer. Reliably identifying the presence and chemical nature of such a trace layer has been challenging. Electron energy-loss spectroscopy (EELS) performed in a scanning transmission electron microscope (STEM), the primary technique utilized for such studies, is limited by a weak signal on overlayers when using conventional analysis methods, often leading to misinterpreted or missed information. Here, a robust, unsupervised machine learning data analysis method is developed to reveal trace encapsulation layers that are otherwise overlooked in STEM-EELS datasets. This method provides a reliable tool for analyzing encapsulation of catalysts and is generally applicable to any spectroscopic analysis of materials and devices where revealing a trace signal and its spatial distribution is challenging.

Optic nerve regeneration screen identifies multiple genes restricting adult neural repair.

Adult mammalian central nervous system (CNS) trauma interrupts neural networks and, because axonal regeneration is minimal, neurological deficits persist. Repair via axonal growth is limited by extracellular inhibitors and cell-autonomous factors. Based on results from a screen in vitro, we evaluate nearly 400 genes through a large-scale in vivo regeneration screen. Suppression of 40 genes using viral-driven short hairpin RNAs (shRNAs) promotes retinal ganglion cell (RGC) axon regeneration after optic nerve crush (ONC), and most are validated by separate CRISPR-Cas9 editing experiments. Expression of these axon-regeneration-suppressing genes is not significantly altered by axotomy. Among regeneration-limiting genes, loss of the interleukin 22 (IL-22) cytokine allows an early, yet transient, inflammatory response in the retina after injury. Reduced IL-22 drives concurrent activation of signal transducer and activator of transcription 3 (Stat3) and dual leucine zipper kinase (DLK) pathways and upregulation of multiple neuron-intrinsic regeneration-associated genes (RAGs). Including IL-22, our screen identifies dozens of genes that limit CNS regeneration. Suppression of these genes in the context of axonal damage could support improved neural repair.

Dynamic morphogenesis of a pioneer axon in Drosophila and its regulation by Abl tyrosine kinase.

The fundamental problem in axon growth and guidance is to understand how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. We here dissect this process using live imaging of the TSM1 axon of the developing Drosophila wing. We find that the growth cone is almost purely filopodial, and that it extends by a protrusive mode of growth. Quantitative analysis reveals two separate groups of growth cone properties that together account for growth cone structure and dynamics. The core morphological features of the growth cone are strongly correlated with one another and define two discrete morphs. Genetic manipulation of a critical mediator of axon guidance signaling, Abelson (Abl) tyrosine kinase, shows that while Abl weakly modulates the ratio of the two morphs it does not greatly change their properties. Rather, Abl primarily regulates the second group of properties, which report the organization and distribution of actin in the growth cone and are coupled to growth cone velocity. Other experiments dissect the nature of that regulation of actin organization and how it controls the spatial localization of filopodial dynamics and thus axon extension. Together, these observations suggest a novel, probabilistic mechanism by which Abl biases the stochastic fluctuations of growth cone actin to direct axon growth and guidance.

Abl signaling directs growth of a pioneer axon in Drosophila by shaping the intrinsic fluctuations of actin.

The fundamental problem in axon growth and guidance is understanding how cytoplasmic signaling modulates the cytoskeleton to produce directed growth cone motility. Live imaging of the TSM1 axon of the developing Drosophila wing has shown that the essential role of the core guidance signaling molecule, Abelson (Abl) tyrosine kinase, is to modulate the organization and spatial localization of actin in the advancing growth cone. Here, we dissect in detail the properties of that actin organization and its consequences for growth cone morphogenesis and motility. We show that advance of the actin mass in the distal axon drives the forward motion of the dynamic filopodial domain that defines the growth cone. We further show that Abl regulates both the width of the actin mass and its internal organization, spatially biasing the intrinsic fluctuations of actin to achieve net advance of the actin, and thus of the dynamic filopodial domain of the growth cone, while maintaining the essential coherence of the actin mass itself. These data suggest a model whereby guidance signaling systematically shapes the intrinsic, stochastic fluctuations of actin in the growth cone to produce axon growth and guidance.

Identification and functional characterization of two novel mutations in KCNJ10 and PI4KB in SeSAME syndrome without electrolyte imbalance.

BACKGROUND: Dysfunction in inwardly rectifying potassium channel Kir4.1 has been implicated in SeSAME syndrome, an autosomal-recessive (AR), rare, multi-systemic disorder. However, not all neurological, intellectual disability, and comorbid phenotypes in SeSAME syndrome can be mechanistically linked solely to Kir4.1 dysfunction. METHODS: We therefore performed whole-exome sequencing and identified additional genetic risk-elements that might exert causative effects either alone or in concert with Kir4.1 in a family diagnosed with SeSAME syndrome. RESULTS: Two variant prioritization pipelines based on AR inheritance and runs of homozygosity (ROH), identified two novel homozygous variants in KCNJ10 and PI4KB and five rare homozygous variants in PVRL4, RORC, FLG2, FCRL1, NIT1 and one common homozygous variant in HSPA6 segregating in all four patients. The novel mutation in KCNJ10 resides in the cytoplasmic domain of Kir4.1, a seat of phosphatidylinositol bisphosphate (PIP2) binding. The mutation altered the subcellular localization and stability of Kir4.1 in patient-specific lymphoblastoid cells (LCLs) compared to parental controls. Barium-sensitive endogenous K+ currents in patient-specific LCLs using whole-cell patch-clamp electrophysiology revealed membrane depolarization and defects in inward K+ ion conductance across the membrane, thereby suggesting a loss-of-function effect of KCNJ10 variant. CONCLUSION: Altogether, our findings implicate the role of new genes in SeSAME syndrome without electrolyte imbalance and thereby speculate the regulation of Kir4.1 channel activity by PIP2 and integrin-mediated adhesion signaling mechanisms.

Chronic Exposure to Chewing Tobacco Induces Metabolic Reprogramming and Cancer Stem Cell-Like Properties in Esophageal Epithelial Cells.

Tobacco in its smoke and smokeless form are major risk factors for esophageal squamous cell carcinoma (ESCC). However, molecular alterations associated with smokeless tobacco exposure are poorly understood. In the Indian subcontinent, tobacco is predominantly consumed in chewing form. An understanding of molecular alterations associated with chewing tobacco exposure is vital for identifying molecular markers and potential targets. We developed an in vitro cellular model by exposing non-transformed esophageal epithelial cells to chewing tobacco over an eight-month period. Chronic exposure to chewing tobacco led to increase in cell proliferation, invasive ability and anchorage independent growth, indicating cell transformation. Molecular alterations associated with chewing tobacco exposure were characterized by carrying out exome sequencing and quantitative proteomic profiling of parental cells and chewing tobacco exposed cells. Quantitative proteomic analysis revealed increased expression of cancer stem cell markers in tobacco treated cells. In addition, tobacco exposed cells showed the Oxidative Phosphorylation (OXPHOS) phenotype with decreased expression of enzymes associated with glycolytic pathway and increased expression of a large number of mitochondrial proteins involved in electron transport chain as well as enzymes of the tricarboxylic acid (TCA) cycle. Electron micrographs revealed increase in number and size of mitochondria. Based on these observations, we propose that chronic exposure of esophageal epithelial cells to tobacco leads to cancer stem cell-like phenotype. These cells show the characteristic OXPHOS phenotype, which can be potentially targeted as a therapeutic strategy.

Does retinoic acid reverse cell cycle dysregulation in Alzheimer's disease lymphocytes?

Aberrant re-entry of neurons into cell cycle appears to be an early event in Alzheimer's disease (AD) and targeting this dysregulation may have therapeutic potential. We have examined whether cell cycle dysregulation in AD can be detected using patient and control derived B-lymphocytes. Cell cycle analysis using flow cytometry demonstrated that cell cycle dysregulation occurs in AD lymphocytes, with a significant difference in the distribution of cells in G0/G1, S and G2/M phases of cell cycle as compared to control lymphocytes. Using global gene expression analysis by RNA sequencing and cell cycle analysis, we examined the role of Retinoic Acid (RA), a candidate molecule predicted to be of therapeutic potential in cell cycle dysregulation associated with AD. CCND1, CCNE2, E2F transcription factors which are known to be dysregulated in AD were among the 32 genes that showed differential expression in response to RA treatment thus suggesting a protective role of RA. However, the cell cycle analysis demonstrated that RA did not reverse the cellular phenotype in AD lymphocytes. This suggests that though RA might have a protective role by influencing the expression of cell cycle genes, it might not be able to arrest abnormal re-entry into cell cycle.

Discovery biology of neuropsychiatric syndromes (DBNS): a center for integrating clinical medicine and basic science.

BACKGROUND: There is emerging evidence that there are shared genetic, environmental and developmental risk factors in psychiatry, that cut across traditional diagnostic boundaries. With this background, the Discovery biology of neuropsychiatric syndromes (DBNS) proposes to recruit patients from five different syndromes (schizophrenia, bipolar disorder, obsessive compulsive disorder, Alzheimer's dementia and substance use disorders), identify those with multiple affected relatives, and invite these families to participate in this study. The families will be assessed: 1) To compare neuro-endophenotype measures between patients, first degree relatives (FDR) and healthy controls., 2) To identify cellular phenotypes which differentiate the groups., 3) To examine the longitudinal course of neuro-endophenotype measures., 4) To identify measures which correlate with outcome, and 5) To create a unified digital database and biorepository. METHODS: The identification of the index participants will occur at well-established specialty clinics. The selected individuals will have a strong family history (with at least another affected FDR) of mental illness. We will also recruit healthy controls without family history of such illness. All recruited individuals (N = 4500) will undergo brief clinical assessments and a blood sample will be drawn for isolation of DNA and peripheral blood mononuclear cells (PBMCs). From among this set, a subset of 1500 individuals (300 families and 300 controls) will be assessed on several additional assessments [detailed clinical assessments, endophenotype measures (neuroimaging- structural and functional, neuropsychology, psychophysics-electroencephalography, functional near infrared spectroscopy, eye movement tracking)], with the intention of conducting repeated measurements every alternate year. PBMCs from this set will be used to generate lymphoblastoid cell lines, and a subset of these would be converted to induced pluripotent stem cell lines and also undergo whole exome sequencing. DISCUSSION: We hope to identify unique and overlapping brain endophenotypes for major psychiatric syndromes. In a proportion of subjects, we expect these neuro-endophenotypes to progress over time and to predict treatment outcome. Similarly, cellular assays could differentiate cell lines derived from such groups. The repository of biomaterials as well as digital datasets of clinical parameters, will serve as a valuable resource for the broader scientific community who wish to address research questions in the area.

Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila, that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons.

New perspectives on the roles of Abl tyrosine kinase in axon patterning.

The Abelson tyrosine kinase (Abl) lies at the heart of one of the small set of ubiquitous, conserved signal transduction pathways that do much of the work of development and physiology. Abl signaling is essential to epithelial integrity, motility of autonomous cells such as blood cells, and axon growth and guidance in the nervous system. However, though Abl was one of the first of these conserved signaling machines to be identified, it has been among the last to have its essential architecture elucidated. Here we will first discuss some of the challenges that long delayed the dissection of this pathway, and what they tell us about the special problems of investigating dynamic processes like motility. We will then describe our recent experiments that revealed the functional organization of the Abl pathway in Drosophila neurons. Finally, in the second part of the review we will introduce a different kind of complexity in the role of Abl in motility: the discovery of a previously unappreciated function in protein secretion and trafficking. We will provide evidence that the secretory function of Abl also contributes to its role in axon growth and guidance, and finally end with a discussion of the challenges that Abl pleiotropy provide for the investigator, but the opportunities that it provides for coordinating biological regulation.

The Abl pathway bifurcates to balance Enabled and Rac signaling in axon patterning in Drosophila.

The Abl tyrosine kinase signaling network controls cell migration, epithelial organization, axon patterning and other aspects of development. Although individual components are known, the relationships among them remain unresolved. We now use FRET measurements of pathway activity, analysis of protein localization and genetic epistasis to dissect the structure of this network in Drosophila We find that the adaptor protein Disabled stimulates Abl kinase activity. Abl suppresses the actin-regulatory factor Enabled, and we find that Abl also acts through the GEF Trio to stimulate the signaling activity of Rac GTPase: Abl gates the activity of the spectrin repeats of Trio, allowing them to relieve intramolecular repression of Trio GEF activity by the Trio N-terminal domain. Finally, we show that a key target of Abl signaling in axons is the WAVE complex that promotes the formation of branched actin networks. Thus, we show that Abl constitutes a bifurcating network, suppressing Ena activity in parallel with stimulation of WAVE. We suggest that the balancing of linear and branched actin networks by Abl is likely to be central to its regulation of axon patterning.

The Abl/enabled signaling pathway regulates Golgi architecture in Drosophila photoreceptor neurons.

The Golgi apparatus is optimized separately in different tissues for efficient protein trafficking, but we know little of how cell signaling shapes this organelle. We now find that the Abl tyrosine kinase signaling pathway controls the architecture of the Golgi complex in Drosophila photoreceptor (PR) neurons. The Abl effector, Enabled (Ena), selectively labels the cis-Golgi in developing PRs. Overexpression or loss of function of Ena increases the number of cis- and trans-Golgi cisternae per cell, and Ena overexpression also redistributes Golgi to the most basal portion of the cell soma. Loss of Abl or its upstream regulator, the adaptor protein Disabled, lead to the same alterations of Golgi as does overexpression of Ena. The increase in Golgi number in Abl mutants arises in part from increased frequency of Golgi fission events and a decrease in fusions, as revealed by live imaging. Finally, we demonstrate that the effects of Abl signaling on Golgi are mediated via regulation of the actin cytoskeleton. Together, these data reveal a direct link between cell signaling and Golgi architecture. Moreover, they raise the possibility that some of the effects of Abl signaling may arise, in part, from alterations of protein trafficking and secretion.

A genome-wide analysis reveals that the Drosophila transcription factor Lola promotes axon growth in part by suppressing expression of the actin nucleation factor Spire.

BACKGROUND: The phylogenetically conserved transcription factor Lola is essential for many aspects of axon growth and guidance, synapse formation and neural circuit development in Drosophila. To date it has been difficult, however, to obtain an overall view of Lola functions and mechanisms. RESULTS: We use expression microarrays to identify the lola-dependent transcriptome in the Drosophila embryo. We find that lola regulates the expression of a large selection of genes that are known to affect each of several lola-dependent developmental processes. Among other loci, we find lola to be a negative regulator of spire, an actin nucleation factor that has been studied for its essential role in oogenesis. We show that spire is expressed in the nervous system and is required for a known lola-dependent axon guidance decision, growth of ISNb motor axons. We further show that reducing spire gene dosage suppresses this aspect of the lola phenotype, verifying that derepression of spire is an important contributor to the axon stalling phenotype of embryonic motor axons in lola mutants. CONCLUSIONS: These data shed new light on the molecular mechanisms of many lola-dependent processes, and also identify several developmental processes not previously linked to lola that are apt to be regulated by this transcription factor. These data further demonstrate that excessive expression of the actin nucleation factor Spire is as deleterious for axon growth in vivo as is the loss of Spire, thus highlighting the need for a balance in the elementary steps of actin dynamics to achieve effective neuronal morphogenesis.

Disabled is a bona fide component of the Abl signaling network.

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.

Abdominal-A mediated repression of Cyclin E expression during cell-fate specification in the Drosophila central nervous system.

Homeotic/Hox genes are known to specify a given developmental pathway by regulating the expression of downstream effector genes. During embryonic CNS development of Drosophila, the Hox protein Abdominal-A (AbdA) is required for the specification of the abdominal NB6-4 lineage. It does so by down regulating the expression of the cell cycle regulator gene Dcyclin E (CycE). CycE is normally expressed in the thoracic NB6-4 lineage to give rise to mixed lineage of neurons and glia, while only glial cells are produced from the abdominal NB6-4 lineage due to the repression of CycE by AbdA. Here we investigate how AbdA represses the expression of CycE to define the abdominal fate of a single NB6-4 precursor cell. We analyze, both in vitro and in vivo, the regulation of a 1.9 kb CNS-specific CycE enhancer element in the abdominal NB6-4 lineage. We show that CycE is a direct target of AbdA and it binds to the CNS specific enhancer of CycE to specifically repress the enhancer activity in vivo. Our results suggest preferential involvement of a series of multiple AbdA binding sites to selectively enhance the repression of CycE transcription. Furthermore, our data suggest a complex network to regulate CycE expression where AbdA functions as a key regulator.