Rational Design of TDP-43 Derived α-Helical Peptide Inhibitors: An In Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders.

TDP-43, an essential RNA/DNA-binding protein, is central to the pathology of neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia. Pathological mislocalization and aggregation of TDP-43 disrupt RNA splicing, mRNA stability, and mRNA transport, thereby impairing neuronal function and survival. The formation of amyloid-like TDP-43 filaments is largely facilitated by the destabilization of an α-helical segment within the disordered C-terminal region. In this study, we hypothesized that preventing the destabilization of the α-helical domain could potentially halt the growth of these pathological filaments. To explore this, we utilized a range of in silico techniques to design and evaluate peptide-based therapeutics that bind to pathological TDP-43 amyloid-like filament crystal structures and resist ß sheet conversion. Our computational approaches, including biophysical and secondary structure property prediction, molecular docking, 3D structure prediction, and molecular dynamics simulations, were used to assess the structure, stability, and binding affinity of these peptides in relation to pathological TDP-43 filaments. The results of our in silico analyses identified a selection of promising peptides which displayed a stable α-helical structure, exhibited an increased number of intramolecular hydrogen bonds within the helical domain, and demonstrated high binding affinities for pathological TDP-43 amyloid-like filaments. Molecular dynamics simulations provided further support for the structural and thermodynamic stability of these peptides, as they exhibited lower root-mean-square deviation and more favorable free energy landscapes over 300 ns. These findings establish α-helical propensity peptides as potential lead molecules for the development of novel therapeutics against TDP-43 aggregation. This structure-based computational approach for the rational design of peptide inhibitors opens a new direction in the search for effective interventions for ALS, FTD, and other related neurodegenerative diseases. The peptides identified as the most promising candidates in this study are currently subject to further testing and validation through both in vitro and in vivo experiments.

Rational Design of TDP-43 Derived α-Helical Peptide Inhibitors: an In-Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders.

TDP-43, an essential RNA/DNA-binding protein, is central to the pathology of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Pathological mislocalization and aggregation of TDP-43 disrupts RNA splicing, mRNA stability, and mRNA transport, thereby impairing neuronal function and survival. The formation of amyloid-like TDP-43 filaments is largely facilitated by the destabilization of an α-helical segment within the disordered C-terminal region. In this study, we hypothesized that preventing the destabilization of the α-helical domain could potentially halt the growth of these pathological filaments. To explore this, we utilized a range of in-silico techniques to design and evaluate peptide-based therapeutics. Various pathological TDP-43 amyloid-like filament crystal structures were selected for their potential to inhibit the binding of additional TDP-43 monomers to the growing filaments. Our computational approaches included biophysical and secondary structure property prediction, molecular docking, 3D structure prediction, and molecular dynamics simulations. Through these techniques, we were able to assess the structure, stability, and binding affinity of these peptides in relation to pathological TDP-43 filaments. The results of our in-silico analyses identified a selection of promising peptides, which displayed a stable α-helical structure, exhibited an increased number of intramolecular hydrogen bonds within the helical domain, and demonstrated high binding affinities for pathological TDP-43 amyloid-like filaments. Additionally, molecular dynamics simulations provided further support for the stability of these peptides, as they exhibited lower root mean square deviations in their helical propensity over 100ns. These findings establish α-helical propensity peptides as potential lead molecules for the development of novel therapeutics against TDP-43 aggregation. This structure-based computational approach for rational design of peptide inhibitors opens a new direction in the search for effective interventions for ALS, FTD, and other related neurodegenerative diseases. The peptides identified as the most promising candidates in this study are currently subject to further testing and validation through both in vitro and in vivo experiments.

Phase separation and pathologic transitions of RNP condensates in neurons: implications for amyotrophic lateral sclerosis, frontotemporal dementia and other neurodegenerative disorders.

Liquid-liquid phase separation results in the formation of dynamic biomolecular condensates, also known as membrane-less organelles, that allow for the assembly of functional compartments and higher order structures within cells. Multivalent, reversible interactions between RNA-binding proteins (RBPs), including FUS, TDP-43, and hnRNPA1, and/or RNA (e.g., RBP-RBP, RBP-RNA, RNA-RNA), result in the formation of ribonucleoprotein (RNP) condensates, which are critical for RNA processing, mRNA transport, stability, stress granule assembly, and translation. Stress granules, neuronal transport granules, and processing bodies are examples of cytoplasmic RNP condensates, while the nucleolus and Cajal bodies are representative nuclear RNP condensates. In neurons, RNP condensates promote long-range mRNA transport and local translation in the dendrites and axon, and are essential for spatiotemporal regulation of gene expression, axonal integrity and synaptic function. Mutations of RBPs and/or pathologic mislocalization and aggregation of RBPs are hallmarks of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. ALS/FTD-linked mutations of RBPs alter the strength and reversibility of multivalent interactions with other RBPs and RNAs, resulting in aberrant phase transitions. These aberrant RNP condensates have detrimental functional consequences on mRNA stability, localization, and translation, and ultimately lead to compromised axonal integrity and synaptic function in disease. Pathogenic protein aggregation is dependent on various factors, and aberrant dynamically arrested RNP condensates may serve as an initial nucleation step for pathologic aggregate formation. Recent studies have focused on identifying mechanisms by which neurons resolve phase transitioned condensates to prevent the formation of pathogenic inclusions/aggregates. The present review focuses on the phase separation of neurodegenerative disease-linked RBPs, physiological functions of RNP condensates, and the pathologic role of aberrant phase transitions in neurodegenerative disease, particularly ALS/FTD. We also examine cellular mechanisms that contribute to the resolution of aberrant condensates in neurons, and potential therapeutic approaches to resolve aberrantly phase transitioned condensates at a molecular level.

Clinical utility of whole-genome DNA methylation profiling as a primary molecular diagnostic assay for central nervous system tumors-A prospective study and guidelines for clinical testing.

Background: Central nervous system (CNS) cancer is the 10th leading cause of cancer-associated deaths for adults, but the leading cause in pediatric patients and young adults. The variety and complexity of histologic subtypes can lead to diagnostic errors. DNA methylation is an epigenetic modification that provides a tumor type-specific signature that can be used for diagnosis. Methods: We performed a prospective study using DNA methylation analysis as a primary diagnostic method for 1921 brain tumors. All tumors received a pathology diagnosis and profiling by whole genome DNA methylation, followed by next-generation DNA and RNA sequencing. Results were stratified by concordance between DNA methylation and histopathology, establishing diagnostic utility. Results: Of the 1602 cases with a World Health Organization histologic diagnosis, DNA methylation identified a diagnostic mismatch in 225 cases (14%), 78 cases (5%) did not classify with any class, and in an additional 110 (7%) cases DNA methylation confirmed the diagnosis and provided prognostic information. Of 319 cases carrying 195 different descriptive histologic diagnoses, DNA methylation provided a definitive diagnosis in 273 (86%) cases, separated them into 55 methylation classes, and changed the grading in 58 (18%) cases. Conclusions: DNA methylation analysis is a robust method to diagnose primary CNS tumors, improving diagnostic accuracy, decreasing diagnostic errors and inconclusive diagnoses, and providing prognostic subclassification. This study provides a framework for inclusion of DNA methylation profiling as a primary molecular diagnostic test into professional guidelines for CNS tumors. The benefits include increased diagnostic accuracy, improved patient management, and refinements in clinical trial design.

Single-cell transcriptomic atlas of Alzheimer's disease middle temporal gyrus reveals region, cell type and sex specificity of gene expression with novel genetic risk for MERTK in female.

Alzheimer's disease, the most common age-related neurodegenerative disease, is closely associated with both amyloid-ß plaque and neuroinflammation. Two thirds of Alzheimer's disease patients are females and they have a higher disease risk. Moreover, women with Alzheimer's disease have more extensive brain histological changes than men along with more severe cognitive symptoms and neurodegeneration. To identify how sex difference induces structural brain changes, we performed unbiased massively parallel single nucleus RNA sequencing on Alzheimer's disease and control brains focusing on the middle temporal gyrus, a brain region strongly affected by the disease but not previously studied with these methods. We identified a subpopulation of selectively vulnerable layer 2/3 excitatory neurons that that were RORB-negative and CDH9-expressing. This vulnerability differs from that reported for other brain regions, but there was no detectable difference between male and female patterns in middle temporal gyrus samples. Disease-associated, but sex-independent, reactive astrocyte signatures were also present. In clear contrast, the microglia signatures of diseased brains differed between males and females. Combining single cell transcriptomic data with results from genome-wide association studies (GWAS), we identified MERTK genetic variation as a risk factor for Alzheimer's disease selectively in females. Taken together, our single cell dataset revealed a unique cellular-level view of sex-specific transcriptional changes in Alzheimer's disease, illuminating GWAS identification of sex-specific Alzheimer's risk genes. These data serve as a rich resource for interrogation of the molecular and cellular basis of Alzheimer's disease.

Ataxin-2 polyglutamine expansions aberrantly sequester TDP-43, drive ribonucleoprotein condensate transport dysfunction and suppress local translation.

Altered RNA metabolism is a common pathogenic mechanism linked to familial and sporadic Amyotrophic lateral sclerosis (ALS). ALS is characterized by mislocalization and aggregation of TDP-43, an RNA-binding protein (RBP) with multiple roles in post-transcriptional RNA processing. Recent studies have identified genetic interactions between TDP-43 and Ataxin-2, a polyglutamine (polyQ) RBP in which intermediate length polyQ expansions confer increased ALS risk. Here, we used live-cell confocal imaging, photobleaching and translation reporter assays to study the localization, transport dynamics and mRNA regulatory functions of TDP-43/Ataxin-2 in rodent primary cortical neurons. We show that Ataxin-2 polyQ expansions aberrantly sequester TDP-43 within ribonucleoprotein (RNP) condensates, and disrupt both its motility along the axon and liquid-like properties. Our data suggest that Ataxin-2 governs motility and translation of neuronal RNP condensates and that Ataxin-2 polyQ expansions fundamentally perturb spatial localization of mRNA and suppress local translation. Overall, these results indicate Ataxin-2 polyQ expansions have detrimental effects on stability, localization, and translation of transcripts critical for axonal and cytoskeletal integrity, particularly important for motor neurons.

Epstein-Barr virus-associated primary intracranial leiomyosarcoma in an immunocompetent patient: illustrative case.

BACKGROUND: Primary intracranial leiomyosarcomas (PILMSs) are extremely rare tumors arising from smooth muscle connective tissue. PILMSs have been shown to be associated with Epstein-Barr virus (EBV). Thus far, EBV-associated PILMS has been exclusively described in immunocompromised patients. OBSERVATIONS: A 40-year-old male presented with a 2-year history of left-sided headaches, nausea, and vomiting. Magnetic resonance imaging demonstrated a large, heterogeneously enhancing, lobulated, dura-based mass arising from the left middle cranial fossa with associated edema and mass effect. The patient underwent an uncomplicated resection of suspected meningioma; neuropathology revealed the exceedingly rare diagnosis of EBV-associated PILMS. Follow-up testing for human immunodeficiency virus (HIV) and other immunodeficiencies confirmed the patient's immunocompetent status. LESSONS: Primary intracranial smooth muscle tumors are often misdiagnosed as meningiomas due to their similar appearance on imaging. PILMSs have a poor prognosis and gross total resection is the mainstay of treatment in the absence of clear recommendations for management. Prompt diagnosis and resection are important; therefore, these tumors should be included in the differential of dura-based tumors, especially among immunocompromised patients. Although EBV-associated PILMSs usually occur in immunocompromised individuals, their presence cannot be ruled out in immunocompetent patients.

Sequence Determinants of TDP-43 Ribonucleoprotein Condensate Formation and Axonal Transport in Neurons.

Mutations in TDP-43, a RNA-binding protein with multiple functions in RNA metabolism, cause amyotrophic lateral sclerosis (ALS), but it is uncertain how defects in RNA biology trigger motor neuron degeneration. TDP-43 is a major constituent of ribonucleoprotein (RNP) granules, phase separated biomolecular condensates that regulate RNA splicing, mRNA transport, and translation. ALS-associated TDP-43 mutations, most of which are found in the low complexity domain, promote aberrant liquid to solid phase transitions and impair the dynamic liquid-like properties and motility of RNP transport granules in neurons. Here, we perform a comparative analysis of ALS-linked mutations and TDP-43 variants in order to identify critical structural elements, aromatic and charged residues that are key determinants of TDP-43 RNP transport and condensate formation in neurons. We find that A315T and Q343R disease-linked mutations and substitutions of aromatic residues within the α-helical domain and LARKS, show the most severe defects in TDP-43 RNP granule transport and impair both anterograde and retrograde motility. F313L and F313-6L/Y substitutions of one or both phenylalanine residues in LARKS suggest the aromatic rings are important for TDP-43 RNP transport. Similarly, W334F/L substitutions of the tryptophan residue in the α-helical domain, impair TDP-43 RNP motility (W334L) or anterograde transport (W334F). We also show that R293A and R293K mutations, which disrupt the only RGG in the LCD, profoundly reduce long-range, directed transport and net velocity of TDP-43 RNP granules. In the disordered regions flanking the α-helical domain, we find that F283Y, F397Y or Y374F substitutions of conserved GF/G and SYS motifs, also impair anterograde and/or retrograde motility, possibly by altering hydrophobicity. Similarly, ALS-linked mutations in disordered regions distant from the α-helical domain also show anterograde transport deficits, consistent with previous findings, but these mutations are less severe than A315T and Q343R. Overall our findings demonstrate that the conserved α-helical domain, phenylalanine residues within LARKS and RGG motif are key determinants of TDP-43 RNP transport, suggesting they may mediate efficient recruitment of motors and adaptor proteins. These results offer a possible mechanism underlying ALS-linked TDP-43 defects in axonal transport and homeostasis.

Comprehensive Genomic Characterization of A Case of Granular Cell Tumor of the Posterior Pituitary Gland: A Case Report.

Granular cell tumors of the pituitary belong to a rare family of neoplasms, arising from the posterior pituitary gland. Although considered benign, they may cause significant morbidity and residual disease after resection can lead to poor clinical outcomes. Currently, there is no known medical therapy for any posterior pituitary gland tumor, in part due to sparse molecular characterization of these lesions. We report data from whole exome sequencing of a case of granular cell tumor of the pituitary, performed under an institutional review board approved protocol. A 77 year-old female underwent resection of an incidentally diagnosed pituitary mass that was causing radiographic compression of the optic nerves with a subclinical temporal field defect and central hypothyroidism. The pathology of the resected specimen demonstrated a granular cell tumor of the posterior pituitary gland. Whole-exome sequencing revealed mutations predicted to be deleterious in key oncogenes, SETD2 and PAX8, both of which have been described in other cancers and could potentially be amenable to targeted therapies with existing approved drugs, including immune checkpoint inhibitors and histone deacetylase inhibitors, respectively. To our knowledge, this is the first comprehensive genomic characterization of granular cell tumor of the posterior pituitary gland. We report mutations in oncogenes predicted to be deleterious and reported in other cancers with potential for therapeutic targeting with existing pharmacologic agents. These data provide new insights into the molecular pathogenesis of GCT of the pituitary and may warrant further investigation.

RNA-binding proteins in neurological development and disease.

RNA-binding proteins are a critical group of multifunctional proteins that precisely regulate all aspects of gene expression, from alternative splicing to mRNA trafficking, stability, and translation. Converging evidence highlights aberrant RNA metabolism as a common pathogenic mechanism in several neurodevelopmental and neurodegenerative diseases. However, dysregulation of disease-linked RNA-binding proteins results in widespread, often tissue-specific and/or pleiotropic effects on the transcriptome, making it challenging to determine the underlying cellular and molecular mechanisms that contribute to disease pathogenesis. Understanding how splicing misregulation as well as alterations of mRNA stability and localization impact the activity and function of neuronal proteins is fundamental to addressing neurodevelopmental defects and synaptic dysfunction in disease. Here we highlight recent exciting studies that use high-throughput transcriptomic analysis and advanced genetic, cell biological, and imaging approaches to dissect the role of disease-linked RNA-binding proteins on different RNA processing steps. We focus specifically on efforts to elucidate the functional consequences of aberrant RNA processing on neuronal morphology, synaptic activity and plasticity in development and disease. We also consider new areas of investigation that will elucidate the molecular mechanisms RNA-binding proteins use to achieve spatiotemporal control of gene expression for neuronal homeostasis and plasticity.

Genetic characterization of an aggressive optic nerve pilocytic glioma.

Optic nerve glioma (ONG) is a rare, typically slow-growing WHO I grade tumor that affects the visual pathways. ONG is most commonly seen in the pediatric population, in association with neurofibromatosis type 1 syndrome. However, sporadic adult cases may also occur and may clinically behave more aggressively, despite benign histopathology. Genetic characterization of these tumors, particularly in the adult population, is lacking. A 39-year-old female presented with 1 month of progressive left-sided visual loss secondary to a enhancing mass along the left optic nerve sheath. Initial empiric management with focal radiotherapy failed to prevent tumor progression, prompting open biopsy which revealed a WHO I pilocytic astrocytoma of the optic nerve. Whole-exome sequencing of the biopsy specimen revealed somatic mutations in NF1,FGFR1 and PTPN11 that may provide actionable targets for molecularly guided therapies. Genetic characterization of ONG is lacking but is needed to guide the management of these rare but complex tumors. The genomic alterations reported in this case contributes to understanding the pathophysiology of adult sporadic ONG and may help guide future clinical prognostication and development of targeted therapies.

Expression of WIPI2B counteracts age-related decline in autophagosome biogenesis in neurons.

Autophagy defects are implicated in multiple late-onset neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's, Huntington's, and Parkinson's diseases. Since aging is the most common shared risk factor in neurodegeneration, we assessed rates of autophagy in mammalian neurons during aging. We identified a significant decrease in the rate of constitutive autophagosome biogenesis during aging and observed pronounced morphological defects in autophagosomes in neurons from aged mice. While early stages of autophagosome formation were unaffected, we detected the frequent production of stalled LC3B-negative isolation membranes in neurons from aged mice. These stalled structures recruited the majority of the autophagy machinery, but failed to develop into LC3B-positive autophagosomes. Importantly, ectopically expressing WIPI2B effectively restored autophagosome biogenesis in aged neurons. This rescue is dependent on the phosphorylation state of WIPI2B at the isolation membrane, suggesting a novel therapeutic target in age-associated neurodegeneration.

Dural Arteriovenous Fistula Associated With a Vestibular Tumor: An Unusual Case and Review of the Literature.

Intracranial dural arteriovenous fistulae (DAVF) are rare vascular malformations. They are generally considered to be acquired lesions, often attributed to dural sinus thrombosis and intracranial venous hypertension. The authors encountered a case of DAVF associated with an octreotide-positive vestibular schwannoma. A 46-year-old female had symptoms of right ear congestion accompanied by pulsatile tinnitus and mild hearing loss. Magnetic resonance imaging (MRI) identified a lobulated mass centered at the cerebellopontine angle. Preoperatively, on cerebral angiography, there was an incidental discovery of a DAVF in the right posterior fossa. The decision was made to proceed with resection of the tumor in a staged fashion. Her latest follow-up MRI showed no evidence of recurrent tumor. This is the second reported case of DAVF associated with an intracranial schwannoma. Findings are discussed along with a thorough review of the literature. This case, combined with the data from the literature review, led us to believe that tumor-related angiogenesis might contribute to DAVF formation.

Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons.

Ribonucleoprotein (RNP) granules are enriched in specific RNAs and RNA-binding proteins (RBPs) and mediate critical cellular processes. Purified RBPs form liquid droplets in vitro through liquid-liquid phase separation and liquid-like non-membrane-bound structures in cells. Mutations in the human RBPs TAR-DNA binding protein 43 (TDP-43) and RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), but the biophysical properties of these proteins have not yet been studied in neurons. Here, we show that TDP-43 RNP granules in axons of rodent primary cortical neurons display liquid-like properties, including fusion with rapid relaxation to circular shape, shear stress-induced deformation, and rapid fluorescence recovery after photobleaching. RNP granules formed from wild-type TDP-43 show distinct biophysical properties depending on axonal location, suggesting maturation to a more stabilized structure is dependent on subcellular context, including local density and aging. Superresolution microscopy demonstrates that the stabilized population of TDP-43 RNP granules in the proximal axon is less circular and shows spiculated edges, whereas more distal granules are both more spherical and more dynamic. RNP granules formed by ALS-linked mutant TDP-43 are more viscous and exhibit disrupted transport dynamics. We propose these altered properties may confer toxic gain of function and reflect differential propensity for pathological transformation.

Advanced neuroimaging findings of pseudotumoral hemicerebellitis in an elderly male requiring surgical decompression.

Acute cerebellitis is an inflammatory process that usually affects bilateral cerebellar hemispheres in the pediatric population. Pseudotumoral hemicerebellitis is an extremely rare presentation in which unilateral cerebellar involvement mimics a tumor that can exert significant mass effect on the surrounding structures, which may require surgical intervention. Magnetic resonance imaging characteristics that suggest cerebellitis include cerebellar swelling, T2 hyperintensity, and pial enhancement. Advanced neuroimaging, including MR perfusion and MR spectroscopy, may be helpful in excluding other diagnoses. The authors present the case of pseudotumoral hemicerebellitis in the oldest documented patient, a 73-year-old man who required surgical decompression, and they provide a brief discussion of advanced neuroimaging findings.

The variable presentations of anaplastic spindle cell squamous carcinoma associated with tall cell variant of papillary thyroid carcinoma.

BACKGROUND: In 1976, Hawk and Hazard described the tall cell variant (TCV) of papillary thyroid carcinoma (PTC). While the lesions they described had cytologic features of papillary carcinoma, they showed more aggressive behavior with a greater propensity for extrathyroid extension and lymphovascular invasion than classic PTC. In 1991, Bronner and LiVolsi described a series of patients with TCV that progressed to spindle cell squamous carcinoma (SCSC), a unique form of anaplastic thyroid carcinoma. This study describes the variable clinical and pathologic presentations in 31 patients with anaplastic SCSC arising in association with TCV. METHODS: The surgical pathology archives as well as the personal consultation files of one of the authors (V.A.L.) were reviewed to retrieve cases of SCSC arising in association with TCV. The available clinical as well as pathologic information on all patients was reviewed. RESULTS: A total of 31 patients with SCSC arising in association with TCV were retrieved from our files. The average age at primary presentation was 67 (range 32-92) with a female-to-male ratio of 2:1. Three clinical scenarios for SCSC associated with TCV were identified. These were type I, consisting of TCV with SCSC at the time of presentation (18 patients); type II, consisting of SCSC arising as a recurrence in patients with a known history of TCV (5 patients); and type III, consisting of SCSC presenting as a primary laryngeal squamous cell carcinoma in a patient with or without a known history of TCV (8 patients). The type III cases were of most concern since they often were confused with primary laryngeal squamous cell carcinoma and most often were diagnosed after laryngectomy. CONCLUSIONS: SCSC of the thyroid is almost exclusively associated with TCV and can have variable clinical presentations. SCSC is most commonly seen associated with a primary diagnosis of TCV. SCSC may be seen, however, in patients with recurrent PTC and most importantly may present in a fashion similar to primary laryngeal SCSC. Therefore, caution should be exercised in evaluating laryngeal squamous lesions in patients with known history of TCV and without known risks factors for head and neck carcinogenesis.

Leading process branch instability in Lis1+/- nonradially migrating interneurons.

Mammalian forebrain development requires extensive migration, yet the mechanisms through which migrating neurons sense and respond to guidance cues are not well understood. Similar to the axon growth cone, the leading process and branches of neurons may guide migration, but the cytoskeletal events that regulate branching are unknown. We have previously shown that loss of microtubule-associated protein Lis1 reduces branching during migration compared with wild-type neurons. Using time-lapse imaging of Lis1(+/-) and Lis1(+/+) cells migrating from medial ganglionic eminence explant cultures, we show that the branching defect is not due to a failure to initiate branches but a defect in the stabilization of new branches. The leading processes of Lis1(+/-) neurons have reduced expression of stabilized, acetylated microtubules compared with Lis1(+/+) neurons. To determine whether Lis1 modulates branch stability through its role as the noncatalytic beta regulatory subunit of platelet-activating factor (PAF) acetylhydrolase 1b, exogenous PAF was applied to wild-type cells. Excess PAF added to wild-type neurons phenocopies the branch instability observed in Lis1(+/-) neurons, and a PAF antagonist rescues leading process branching in Lis1(+/-) neurons. These data highlight a role for Lis1, acting through the PAF pathway, in leading process branching and microtubule stabilization.