Microglia, Trem2, and Neurodegeneration.

Microglia are a specialized type of neuroimmune cells that undergo morphological and molecular changes through multiple signaling pathways in response to pathological protein aggregates, neuronal death, tissue injury, or infections. Microglia express Trem2, which serves as a receptor for a multitude of ligands enhancing their phagocytic activity. Trem2 has emerged as a critical modulator of microglial activity, especially in many neurodegenerative disorders. Human TREM2 mutations are associated with an increased risk of developing Alzheimer disease (AD) and other neurodegenerative diseases. Trem2 plays dual roles in neuroinflammation and more specifically in disease-associated microglia. Most recent developments on the molecular mechanisms of Trem2, emphasizing its role in uptake and clearance of amyloid ß (Aß) aggregates and other tissue debris to help protect and preserve the brain, are encouraging. Although Trem2 normally stimulates defense mechanisms, its dysregulation can intensify inflammation, which poses major therapeutic challenges. Recent therapeutic approaches targeting Trem2 via agonistic antibodies and gene therapy methodologies present possible avenues for reducing the burden of neurodegenerative diseases. This review highlights the promise of Trem2 as a therapeutic target, especially for Aß-associated AD, and calls for more mechanistic investigations to understand the context-specific role of microglial Trem2 in developing effective therapies against neurodegenerative diseases.

Protocol for isolating and processing mouse sciatic nerve fibers for confocal immunohistochemistry.

Many motor and neurodegenerative diseases affect the peripheral nervous system (PNS). The myelinated axons in the sciatic nerves offer valuable insights into the pathology of these diseases. Here, we present a protocol for isolating and processing mouse sciatic nerves for confocal immunohistochemistry. We describe steps for mouse perfusion, removing and fixing the sciatic nerve, transferring nerves onto slides, staining, and imaging. This protocol can assist in characterizing pathologies of myelinated fibers resulting from diseases affecting the PNS. For complete details on the use and execution of this protocol, please refer to Chang et al. (2023).1.

Mouse models of human CNTNAP1-associated congenital hypomyelinating neuropathy and genetic restoration of murine neurological deficits.

The Contactin-associated protein 1 (Cntnap1) mouse mutants fail to establish proper axonal domains in myelinated axons. Human CNTNAP1 mutations are linked to hypomyelinating neuropathy-3, which causes severe neurological deficits. To understand the human neuropathology and to model human CNTNAP1C323R and CNTNAP1R764C mutations, we generated Cntnap1C324R and Cntnap1R765C mouse mutants, respectively. Both Cntnap1 mutants show weight loss, reduced nerve conduction, and progressive motor dysfunction. The paranodal ultrastructure shows everted myelin loops and the absence of axo-glial junctions. Biochemical analysis reveals that these Cntnap1 mutant proteins are nearly undetectable in the paranodes, have reduced surface expression and stability, and are retained in the neuronal soma. Postnatal transgenic expression of Cntnap1 in the mutant backgrounds rescues the phenotypes and restores the organization of axonal domains with improved motor function. This study uncovers the mechanistic impact of two human CNTNAP1 mutations in a mouse model and provides proof of concept for gene therapy for CNTNAP1 patients.

Increasing the Safety Profile of Follicular Unit Extraction by Eliminating the Use of Bupivacaine and Nerve Block: Experience from a Tertiary Care Center of North India.

Introduction: Follicular unit extraction (FUE) is a safe and effective procedure in the hands of an expert. Side effects, particularly those which can lead to significant morbidity or mortality, are unacceptable as the procedure is done purely for cosmetic reasons. Any modification that decreases the risk associated with the procedure should be promoted. Aim and Objective: The study was conducted to determine whether FUE can be carried out effectively with the elimination of nerve blocks and bupivacaine from the procedure. Materials and Methods: The study was conducted in 30 patients suffering from androgenetic alopecia. The donor areas was anesthetized using lignocaine with adrenaline just below the area to be harvested. The anesthetic was injected intradermally resulting in the development of wheals in continuity, forming a linear line. From our previous experience, we found intradermal administration of lignocaine to give better anesthetic effect as compared to subcutaneous administration, although the former is more painful. This was followed by injection of tumescent into the donor area and donor harvesting, which lasted for a couple of hours. The recipient area was anesthetized using a similar technique of linear injection of anesthetic just ahead of the proposed hair line. Results: The total amount of lignocaine with adrenaline consumed during the surgery ranged from a minimum of 6.1 ml to 8.5 ml, with an average of 7.6 ml. The average duration of the entire surgery was 6.5 h, ranging from 4.5 to 8.5 h. None of the patients experienced any pain during the entire surgery, and there were no significant side effects related to anesthetic administration in any patient. Discussion: We found lignocaine with adrenaline to be a very safe and effective anesthetic agent for field block anesthesia in FUE. The exclusion of bupivacaine and nerve blocks from the procedure of FUE can further increase the safety of the procedure, particularly for beginners and in cases where the area to be covered is not extensive (Norwood-Hamilton grades 3, 4, and 5).

Dermoscopy of Oral Mucosal Lesions: Experience from a Tertiary Care Center in North India and Review of Literature.

Background: Patients with mucosal lesions form a significant number of routine outpatients presenting to the dermatology department where diagnostic confirmation using histopathological examination of mucosal biopsy is neither feasible nor warranted in every patient. Objective: To study the dermoscopic features of various mucosal lesions affecting the oral cavity and to assess the reliability of mucoscopy vis-a-vis clinico-laboratory findings. Materials and Methods: An observational, cross-sectional, hospital-based study was conducted over a period of 2 years from March 2019 to February 2021 in the dermatology outpatient department. Patients presenting with oral mucosal lesions, with or without associated cutaneous involvement, were recruited for mucoscopic evaluation after taking an informed written consent. A detailed history and clinical examination, with emphasis on mucocutaneous examination, was performed and findings were recorded on a standard predesigned proforma. Mucoscopy of oral mucosa was carried out using a handheld dermoscope as well as Universal Serial Bus connected video-dermoscope in both nonpolarized and polarized modes. The different mucoscopic features were seen at these sites, compared with each other, analyzed and findings were recorded. A diagnosis was made on the basis of mucoscopic findings and correlated with clinical diagnosis. The data was analyzed using appropriate statistical tests. Results: The mean age of patients was 34.3 years and the mean lesional duration was 68.2 weeks. Oral lichen planus (18.66%) was the most common disorder studied, followed by recurrent apthous stomatitis (16.00%), pigmentary lesions (12.66%), vascular disorders (12.00%), mucocele (5.33%), pemphigus vulgaris (4.66%), and discoid lupus erythematosus (4.66%). Conclusion: Dermoscopy in oral lesions facilitates the visualization of the mucosal surface and provides quick confirmation of diagnosis in various mucosal disorders with advanced diagnostic accuracy. Mucoscopy was found helpful in differentiating the oral ulcers, which are a presenting feature of various serious mucocutaneous disorders. Mucoscopy could be a helpful aid in diagnosing pigmented skin lesions and alleviating the apprehension regarding oral melanoma and serve as a screening tool in case of squamous cell carcinoma lips. Limitations: Confirmatory histopathological analysis and correlation with mucoscopic findings could not be established in our study.

Drosophila Homolog of the Human Carpenter Syndrome Linked Gene, MEGF8, Is Required for Synapse Development and Function.

Drosophila multiple epidermal growth factor-like domains 8 (dMegf8) is a homolog of human MEGF8 MEGF8 encodes a multidomain transmembrane protein which is highly conserved across species. In humans, MEGF8 mutations cause a rare genetic disorder called Carpenter syndrome, which is frequently associated with abnormal left-right patterning, cardiac defects, and learning disabilities. MEGF8 is also associated with psychiatric disorders. Despite its clinical relevance, MEGF8 remains poorly characterized; and although it is highly conserved, studies on animal models of Megf8 are also very limited. The presence of intellectual disabilities in Carpenter syndrome patients and association of MEGF8 with psychiatric disorders indicate that mutations in MEGF8 cause underlying defects in synaptic structure and functions. In this study, we investigated the role of Drosophila dMegf8 in glutamatergic synapses of the larval neuromuscular junctions (NMJ) in both males and females. We show that dMegf8 localizes to NMJ synapses and is required for proper synaptic growth. dMegf8 mutant larvae and adults show severe motor coordination deficits. At the NMJ, dMegf8 mutants show altered localization of presynaptic and postsynaptic proteins, defects in synaptic ultrastructure, and neurotransmission. Interestingly, dMegf8 mutants have reduced levels of the Type II BMP receptor Wishful thinking (Wit). dMegf8 displays genetic interactions with neurexin-1 (dnrx) and wit, and in association with Dnrx and Wit plays an essential role in synapse organization. Our studies provide insights into human MEGF8 functions and potentially into mechanisms that may underlie intellectual disabilities observed in Carpenter syndrome as well as MEGF8-related synaptic structural and/or functional deficits in psychiatric disorders.SIGNIFICANCE STATEMENT Carpenter syndrome, known for over a century now, is a genetic disorder linked to mutations in Multiple Epidermal Growth Factor-like Domains 8 (MEGF8) gene and associated with intellectual disabilities among other symptoms. MEGF8 is also associated with psychiatric disorders. Despite the high genetic conservation and clinical relevance, the functions of MEGF8 remain largely uncharacterized. Patients with intellectual disabilities and psychiatric diseases often have an underlying defect in synaptic structure and function. This work defines the role of the fly homolog of human MEGF8, dMegf8, in glutamatergic synapse growth, organization, and function and provide insights into potential functions of MEGF8 in human central synapses and synaptic mechanisms that may underlie psychiatric disorders and intellectual disabilities seen in Carpenter syndrome.

Microglial mTOR Activation Upregulates Trem2 and Enhances ß-Amyloid Plaque Clearance in the 5XFAD Alzheimer's Disease Model.

The mechanistic target of rapamycin (mTOR) signaling pathway plays a major role in key cellular processes including metabolism and differentiation; however, the role of mTOR in microglia and its importance in Alzheimer's disease (AD) have remained largely uncharacterized. We report that selective loss of Tsc1, a negative regulator of mTOR, in microglia in mice of both sexes, caused mTOR activation and upregulation of Trem2 with enhanced ß-Amyloid (Aß) clearance, reduced spine loss, and improved cognitive function in the 5XFAD AD mouse model. Combined loss of Tsc1 and Trem2 in microglia led to reduced Aß clearance and increased Aß plaque burden revealing that Trem2 functions downstream of mTOR. Tsc1 mutant microglia showed increased phagocytosis with upregulation of CD68 and Lamp1 lysosomal proteins. In vitro studies using Tsc1-deficient microglia revealed enhanced endocytosis of the lysosomal tracker indicator Green DND-26 suggesting increased lysosomal activity. Incubation of Tsc1-deficient microglia with fluorescent-labeled Aß revealed enhanced Aß uptake and clearance, which was blunted by rapamycin, an mTOR inhibitor. In vivo treatment of mice of relevant genotypes in the 5XFAD background with rapamycin, affected microglial activity, decreased Trem2 expression and reduced Aß clearance causing an increase in Aß plaque burden. Prolonged treatment with rapamycin caused even further reduction of mTOR activity, reduction in Trem2 expression, and increase in Aß levels. Together, our findings reveal that mTOR signaling in microglia is critically linked to Trem2 regulation and lysosomal biogenesis, and that the upregulation of Trem2 in microglia through mTOR activation could be exploited toward better therapeutic avenues to Aß-related AD pathologies.SIGNIFICANCE STATEMENT Mechanistic target of rapamycin (mTOR) signaling pathway is a key regulator for major cellular metabolic processes. However, the link between mTOR signaling and Alzheimer's disease (AD) is not well understood. In this study, we provide compelling in vivo evidence that mTOR activation in microglia would benefit ß-Amyloid (Aß)-related AD pathologies, as it upregulates Trem2, a key receptor for Aß plaque uptake. Inhibition of mTOR pathway with rapamycin, a well-established immunosuppressant, downregulated Trem2 in microglia and reduced Aß plaque clearance indicating that mTOR inactivation may be detrimental in Aß-associated AD patients. This finding will have a significant public health impact and benefit, regarding the usage of rapamycin in AD patients, which we believe will aggravate the Aß-related AD pathologies.

Tbx1, a gene encoded in 22q11.2 copy number variant, is a link between alterations in fimbria myelination and cognitive speed in mice.

Copy number variants (CNVs) have provided a reliable entry point to identify the structural correlates of atypical cognitive development. Hemizygous deletion of human chromosome 22q11.2 is associated with impaired cognitive function; however, the mechanisms by which the CNVs contribute to cognitive deficits via diverse structural alterations in the brain remain unclear. This study aimed to determine the cellular basis of the link between alterations in brain structure and cognitive functions in mice with a heterozygous deletion of Tbx1, one of the 22q11.2-encoded genes. Ex vivo whole-brain diffusion-tensor imaging (DTI)-magnetic resonance imaging (MRI) in Tbx1 heterozygous mice indicated that the fimbria was the only region with significant myelin alteration. Electron microscopic and histological analyses showed that Tbx1 heterozygous mice exhibited an apparent absence of large myelinated axons and thicker myelin in medium axons in the fimbria, resulting in an overall decrease in myelin. The fimbria of Tbx1 heterozygous mice showed reduced mRNA levels of Ng2, a gene required to produce oligodendrocyte precursor cells. Moreover, postnatal progenitor cells derived from the subventricular zone, a source of oligodendrocytes in the fimbria, produced fewer oligodendrocytes in vitro. Behavioral analyses of these mice showed selectively slower acquisition of spatial memory and cognitive flexibility with no effects on their accuracy or sensory or motor capacities. Our findings provide a genetic and cellular basis for the compromised cognitive speed in patients with 22q11.2 hemizygous deletion.

Clinical Pattern and Patch Test Profile of Hand Eczema in Hospital Employees in a Tertiary Care Hospital of North India.

INTRODUCTION: Health care workers form an important occupational group with a high risk of hand eczema. All health care professionals are exposed to a variety of allergens and irritants which can cause hand dermatitis, resulting in significant morbidity. AIMS AND OBJECTIVES: To assess the clinical profile of hand eczema in hospital employees, to perform patch test in relevant cases and to find out the most common sensitizers in them. MATERIALS AND METHODS: This was a cross-sectional, hospital-based study in which the staff was screened for features of hand eczema and patch testing was done in the suspected cases of allergic contact dermatitis. RESULTS: Out of 340 employees screened, 46 employees (13.5%) suffered from hand eczema. The most common type was wear and tear dermatitis accounting for 17 (36.9%) cases, followed by discoid eczema, pompholyx, focal palmar peeling, finger-tip eczema, hyperkeratotic eczema, ring eczema, and unspecified types. Patch testing was positive in 15 (32.6%) cases. The most common allergen was paraphenylene diamine, followed by fragrance mix, nitrofurazone, mercaptobenzothiazole, potassium bichromate, black rubber mix, and thiuram mix. A statistically significant association (0.001) was found with an underlying history of atopy. CONCLUSION: Hand eczema is a commonly encountered dermatological complaint in many hospital employees. Proper counseling, work, up, patch testing, and treatment can mitigate the symptoms in such employees.

Ethanol abolishes vigilance-dependent astroglia network activation in mice by inhibiting norepinephrine release.

Norepinephrine adjusts sensory processing in cortical networks and gates plasticity enabling adaptive behavior. The actions of norepinephrine are profoundly altered by recreational drugs like ethanol, but the consequences of these changes on distinct targets such as astrocytes, which exhibit norepinephrine-dependent Ca2+ elevations during vigilance, are not well understood. Using in vivo two-photon imaging, we show that locomotion-induced Ca2+ elevations in mouse astroglia are profoundly inhibited by ethanol, an effect that can be reversed by enhancing norepinephrine release. Vigilance-dependent astroglial activation is abolished by deletion of α1A-adrenergic receptor from astroglia, indicating that norepinephrine acts directly on these ubiquitous glial cells. Ethanol reduces vigilance-dependent Ca2+ transients in noradrenergic terminals, but has little effect on astroglial responsiveness to norepinephrine, suggesting that ethanol suppresses their activation by inhibiting norepinephrine release. Since abolition of astroglia Ca2+ activation does not affect motor coordination, global suppression of astroglial networks may contribute to the cognitive effects of alcohol intoxication.

Focal loss of the paranodal domain protein Neurofascin155 in the internal capsule impairs cortically induced muscle activity in vivo.

Paranodal axoglial junctions are essential for rapid nerve conduction and the organization of axonal domains in myelinated axons. Neurofascin155 (Nfasc155) is a glial cell adhesion molecule that is also required for the assembly of these domains. Previous studies have demonstrated that general ablation of Nfasc155 disorganizes these domains, reduces conduction velocity, and disrupts motor behaviors. Multiple sclerosis (MS), a typical disorder of demyelination in the central nervous system, is reported to have autoantibody to Nfasc. However, the impact of focal loss of Nfasc155, which may occur in MS patients, remains unclear. Here, we examined whether restricted focal loss of Nfasc155 affects the electrophysiological properties of the motor system in vivo. Adeno-associated virus type5 (AAV5) harboring EGFP-2A-Cre was injected into the glial-enriched internal capsule of floxed-Neurofascin (NfascFlox/Flox) mice to focally disrupt paranodal junctions in the cortico-fugal fibers from the motor cortex to the spinal cord. Electromyograms (EMGs) of the triceps brachii muscles in response to electrical stimulation of the motor cortex were successively examined in these awake mice. EMG analysis showed significant delay in the onset and peak latencies after AAV injection compared to control (Nfasc+/+) mice. Moreover, EMG half-widths were increased, and EMG amplitudes were gradually decreased by 13 weeks. Similar EMG changes have been reported in MS patients. These findings provide physiological evidence that motor outputs are obstructed by focal ablation of paranodal junctions in myelinated axons. Our findings may open a new path toward development of a novel biomarker for an early phase of human MS, as Nfasc155 detects microstructural changes in the paranodal junction.

Interplay between RNA-binding protein HuR and Nox4 as a novel therapeutic target in diabetic kidney disease.

OBJECTIVE: Glomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology. METHODS: We studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice. RESULTS: We demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3' untranslated region (3'-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of invitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3'-UTR in response to HG. The invivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function. CONCLUSIONS: We established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.

Ablation of cytoskeletal scaffolding proteins, Band 4.1B and Whirlin, leads to cerebellar purkinje axon pathology and motor dysfunction.

The cerebellar cortex receives neural information from other brain regions to allow fine motor coordination and motor learning. The primary output neurons from the cerebellum are the Purkinje neurons that transmit inhibitory responses to deep cerebellar nuclei through their myelinated axons. Altered morphological organization and electrical properties of the Purkinje axons lead to detrimental changes in locomotor activity often leading to cerebellar ataxias. Two cytoskeletal scaffolding proteins Band 4.1B (4.1B) and Whirlin (Whrn) have been previously shown to play independent roles in axonal domain organization and maintenance in myelinated axons in the spinal cord and sciatic nerves. Immunoblot analysis had indicated cerebellar expression for both 4.1B and Whrn; however, their subcellular localization and cerebellum-specific functions have not been characterized. Using 4.1B and Whrn single and double mutant animals, we show that both proteins are expressed in common cellular compartments of the cerebellum and play cooperative roles in preservation of the integrity of Purkinje neuron myelinated axons. We demonstrate that both 4.1B and Whrn are required for the maintenance of axonal ultrastructure and health. Loss of 4.1B and Whrn leads to axonal transport defects manifested by formation of swellings containing cytoskeletal components, membranous organelles, and vesicles. Moreover, ablation of both proteins progressively affects cerebellar function with impairment in locomotor performance detected by altered gait parameters. Together, our data indicate that 4.1B and Whrn are required for maintaining proper axonal cytoskeletal organization and axonal domains, which is necessary for cerebellum-controlled fine motor coordination.

mTORC1 Activation by Loss of Tsc1 in Myelinating Glia Causes Downregulation of Quaking and Neurofascin 155 Leading to Paranodal Domain Disorganization.

Mutations in human tuberous sclerosis complex (TSC) genes TSC1 and TSC2 are the leading causes of developmental brain abnormalities and large tumors in other tissues. Murine Tsc1/2 have been shown to negatively regulate the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in most tissues, and this pathway has been shown to be essential for proper oligodendrocytes/Schwann cell differentiation and myelination. Here, we report that ablation of Tsc1 gene specifically in oligodendrocytes/Schwann cells activates mTORC1 signaling resulting in severe motor disabilities, weight loss, and early postnatal death. The mutant mice of either sex showed reduced myelination, disrupted paranodal domains in myelinated axons, and disorganized unmyelinated Remak bundles. mRNA and protein expression analyses revealed strong reduction in the RNA-binding protein Quaking (Qk) and the 155 kDa glial Neurofascin (NfascNF155). Re-introduction of exogenous Qk gene in Tsc1 mutant oligodendrocytes restored NfascNF155 protein levels indicating that Qk is required for the stabilization of NfascNF155 mRNA. Interestingly, injection of Rapamycin, a pharmacological mTORC1 inhibitor, to pregnant mothers increased the lifespan of the mutant offspring, restored myelination as well as the levels of Qk and NfascNF155, and consequently the organization of the paranodal domains. Together our studies show a critical role of mTORC1 signaling in the differentiation of myelinating glial cells and proper organization of axonal domains and provide insights into TSC-associated myelinated axon abnormalities.

Dysregulation of schizophrenia-related aquaporin 3 through disruption of paranode influences neuronal viability.

Myelinated axons segregate the axonal membrane into four defined regions: the node of Ranvier, paranode, juxtaparanode, and internode. The paranodal junction consists of specific component proteins, such as neurofascin155 (NF155) on the glial side, and Caspr and Contactin on the axonal side. Although paranodal junctions are thought to play crucial roles in rapid saltatory conduction and nodal assembly, the role of their interaction with neurons is not fully understood. In a previous study, conditional NF155 knockout in oligodendrocytes led to disorganization of the paranodal junctions. To examine if disruption of paranodal junctions affects neuronal gene expression, we prepared total RNA from the retina of NF155 conditional knockout, and performed expression analysis. We found that the expression level of 433 genes changed in response to paranodal junction ablation. Interestingly, expression of aquaporin 3 (AQP3) was significantly reduced in NF155 conditional knockout mice, but not in cerebroside sulfotransferase knockout (CST-KO) mice, whose paranodes are not originally formed during development. Copy number variations have an important role in the etiology of schizophrenia (SCZ). We observed rare duplications of AQP3 in SCZ patients, suggesting a correlation between abnormal AQP3 expression and SCZ. To determine if AQP3 over-expression in NF155 conditional knockout mice influences neuronal function, we performed adeno-associated virus (AAV)-mediated over-expression of AQP3 in the motor cortex of mice and found a significant increase in caspase 3-dependent neuronal apoptosis in AQP3-transduced cells. This study may provide new insights into therapeutic approaches for SCZ by regulating AQP3 expression, which is associated with paranodal disruption.

Simultaneous Ablation of Neuronal Neurofascin and Ankyrin G in Young and Adult Mice Reveals Age-Dependent Increase in Nodal Stability in Myelinated Axons and Differential Effects on the Lifespan.

Nodes of Ranvier are unique regions where voltage-gated sodium channels are highly enriched to drive saltatory conduction. Genetic ablations in adult mice with loss of specific nodal proteins causes slow but progressive nodal deterioration associated with decreased nerve conduction and axonopathy. What has remained unaddressed is whether loss of nodal proteins at different time points in postnatal life follows similar timelines of nodal disorganization. Here we utilized simultaneous ablation of Neurofascin (NF186) and Ankyrin G (AnkG) in mice of both sexes at three specific time points. We report that concurrent ablation of these core nodal components at postnatal day 13 (P13) leads to accelerated nodal destabilization in comparison with P23, and this disorganization is even slower when ablated at P93. Ablation of NF186 with AnkG at P13 reduced the half-life of NF186 to 15 days compared to 1 month at P23, which increased to 2 months at P93, indicating increasing nodal stability. The half-life of AnkG at the nodes also increased with age but showed enhanced disappearance from the node in the absence of NF186, with a half-life of 3 days at P13 ablation. The nodal disorganization occurred in a sequential manner, with AnkG disappearing first from the nodal areas irrespective of the timing of ablation, and led to decreased nerve conduction and affected axonal health. Together, our studies reveal that nodes of Ranvier in myelinated axons continue to become more stable with age and suggest that nodal disorganization in adult human demyelinating disorders occurs slowly until neurological symptoms become evident.

Reorganization of Destabilized Nodes of Ranvier in ßIV Spectrin Mutants Uncovers Critical Timelines for Nodal Restoration and Prevention of Motor Paresis.

Disorganization of nodes of Ranvier is associated with motor and sensory dysfunctions. Mechanisms that allow nodal recovery during pathological processes remain poorly understood. A highly enriched nodal cytoskeletal protein ßIV spectrin anchors and stabilizes the nodal complex to actin cytoskeleton. Loss of murine ßIV spectrin allows the initial nodal organization, but causes gradual nodal destabilization. Mutations in human ßIV spectrin cause auditory neuropathy and impairment in motor coordination. Similar phenotypes are caused by nodal disruption due to demyelination. Here we report on the precise timelines of nodal disorganization and reorganization by following disassembly and reassembly of key nodal proteins in ßIV spectrin mice of both sexes before and after ßIV spectrin re-expression at specifically chosen developmental time points. We show that the timeline of nodal restoration has different outcomes in the PNS and CNS with respect to nodal reassembly and functional restoration. In the PNS, restoration of nodes occurs within 1 month regardless of the time of ßIV spectrin re-expression. In contrast, the CNS nodal reorganization and functional restoration occurs within a critical time window; after that, nodal reorganization diminishes, leading to less efficient motor recovery. We demonstrate that timely restoration of nodes can improve both the functional properties and the ultrastructure of myelinated fibers affected by long-term nodal disorganization. Our studies, which indicate a critical timeline for nodal restoration together with overall motor performance and prolonged life span, further support the idea that nodal restoration is more beneficial if initiated before any axonal damage, which is critically relevant to demyelinating disorders.SIGNIFICANCE STATEMENT Nodes of Ranvier are integral to efficient and rapid signal transmission along myelinated fibers. Various demyelinating disorders are characterized by destabilization of the nodal molecular complex, accompanied by severe reduction in nerve conduction and the onset of motor and sensory dysfunctions. This study is the first to report in vivo reassembly of destabilized nodes with sequential improvement in overall motor performance. Our study reveals that nodal restoration is achievable before any axonal damage, and that long-term nodal destabilization causes irreversible axonal structural changes that prevent functional restoration. Our studies provide significant insights into timely restoration of nodal domains as a potential therapeutic approach in treatment of demyelinating disorders.

Nox4 is a Target for Tuberin Deficiency Syndrome.

The mechanism by which TSC2 inactivation or deficiency contributes to the pathology of tuberous sclerosis complex (TSC) is not fully clear. We show that renal angiomyolipomas from TSC patients and kidney cortex from Tsc2+/- mice exhibit elevated levels of reactive oxygen species (ROS). Downregulation of tuberin (protein encoded by TSC2 gene) in renal proximal tubular epithelial cells significantly increased ROS concomitant with enhanced Nox4. Similarly, we found elevated levels of Nox4 in the renal cortex of Tsc2+/- mice and in the renal angiomyolipomas from TSC patients. Tuberin deficiency is associated with activation of mTORC1. Rapamycin, shRNAs targeting raptor, or inhibition of S6 kinase significantly inhibited the expression of Nox4, resulting in attenuation of production of ROS in tuberin-downregulated proximal tubular epithelial cells. In contrast, activation of mTORC1 increased Nox4 and ROS. These results indicate that Nox4 may be a potential target for tuberin-deficiency-derived diseases. Using a xenograft model from tuberin-null tubular cells in nude mice, both anti-sense Nox4 and GKT137831, a specific inhibitor of Nox1/4, significantly inhibited the tumor growth. Thus, our results demonstrate the presence of an antagonistic relationship between tuberin and Nox4 to drive oncogenesis in the tuberin deficiency syndrome and identify Nox4 as a target to develop a therapy for TSC.

Myelination of Purkinje axons is critical for resilient synaptic transmission in the deep cerebellar nucleus.

The roles of myelin in maintaining axonal integrity and action potential (AP) propagation are well established, but its role in synapse maintenance and neurotransmission remains largely understudied. Here, we investigated how Purkinje axon myelination regulates synaptic transmission in the Purkinje to deep cerebellar nuclei (DCN) synapses using the Long Evans Shaker (LES) rat, which lacks compact myelin and thus displays severe locomotion deficits. DCN neurons fired spontaneous action potentials (APs), whose frequencies were dependent on the extent of myelin. In the LES cerebellum with severe myelin deficiency, DCN neurons were hyper-excitable, exhibiting spontaneous AP firing at a much higher frequency compared to those from wild type (LE) and heterozygote (LEHet) rats. The hyper-excitability in LES DCN neurons resulted from reduced inhibitory GABAergic inputs from Purkinje cells to DCN neurons. Corresponding with functional alterations including failures of AP propagation, electron microscopic analysis revealed anatomically fewer active zones at the presynaptic terminals of Purkinje cells in both LEHet and LES rats. Taken together, these studies suggest that proper axonal myelination critically regulates presynaptic terminal structure and function and directly impacts synaptic transmission in the Purkinje cell-DCN cell synapse in the cerebellum.

A versatile genetic tool to study midline glia function in the Drosophila CNS.

Neuron-glial interactions are crucial for growth, guidance and ensheathment of axons across species. In the Drosophila CNS midline, neuron-glial interactions underlie ensheathment of commissural axons by midline glial (MG) cells in a manner similar to mammalian oligodendrocytes. Although there has been some advance in the study of neuron-glial interactions and ensheathment of axons in the CNS midline, key aspects of axonal ensheathment are still not fully understood. One of the limitations has been the unavailability of MG membrane markers that could highlight the glial processes wrapping the axons. Previous studies have identified two key molecular players from the neuronal and glial cell types in the CNS midline. These are the neuronal transmembrane protein Neurexin IV (Nrx IV) and the membrane-anchored MG protein Wrapper, both of which interact in trans to mediate neuron-glial interactions and ensheathment of commissural axons. In the current study, we attempt to further our understanding of MG biology and try to overcome some of the technical difficulties posed by the lack of a robust MG driver that will specifically allow expression or knockdown of genes in MG. We report the generation of BAC transgenic flies of wrapper-GAL4 and demonstrate how these flies could be used as a genetic tool to understand MG biology. We have utilized the GAL4/UAS system to drive GFP-reporter lines (membrane-bound mCD8-GFP; microtubule-associated tau-GFP) and nuclear lacZ using wrapper-GAL4 to highlight the MG cells and/or their processes that surround and perform axonal ensheathment functions in the embryonic midline. We also describe the utility of the wrapper-GAL4 driver line to down-regulate known MG genes specifically in Wrapper-positive cells. Finally, we validate the functionality of the wrapper-GAL4 driver by rescue of wrapper mutant phenotypes and lethality. Together, these studies provide us with a versatile genetic tool to investigate MG functions and will aid in future investigations where genetic screens using wrapper-GAL4 could be designed to identify novel molecular players at the Drosophila midline and unravel key aspects of MG biology.