Deep sequencing of proteotoxicity modifier genes uncovers a Presenilin-2/beta-amyloid-actin genetic risk module shared among alpha-synucleinopathies.

Whether neurodegenerative diseases linked to misfolding of the same protein share genetic risk drivers or whether different protein-aggregation pathologies in neurodegeneration are mechanistically related remains uncertain. Conventional genetic analyses are underpowered to address these questions. Through careful selection of patients based on protein aggregation phenotype (rather than clinical diagnosis) we can increase statistical power to detect associated variants in a targeted set of genes that modify proteotoxicities. Genetic modifiers of alpha-synuclein (ɑS) and beta-amyloid (Aß) cytotoxicity in yeast are enriched in risk factors for Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. Here, along with known AD/PD risk genes, we deeply sequenced exomes of 430 ɑS/Aß modifier genes in patients across alpha-synucleinopathies (PD, Lewy body dementia and multiple system atrophy). Beyond known PD genes GBA1 and LRRK2, rare variants AD genes (CD33, CR1 and PSEN2) and Aß toxicity modifiers involved in RhoA/actin cytoskeleton regulation (ARGHEF1, ARHGEF28, MICAL3, PASK, PKN2, PSEN2) were shared risk factors across synucleinopathies. Actin pathology occurred in iPSC synucleinopathy models and RhoA downregulation exacerbated ɑS pathology. Even in sporadic PD, the expression of these genes was altered across CNS cell types. Genome-wide CRISPR screens revealed the essentiality of PSEN2 in both human cortical and dopaminergic neurons, and PSEN2 mutation carriers exhibited diffuse brainstem and cortical synucleinopathy independent of AD pathology. PSEN2 contributes to a common-risk signal in PD GWAS and regulates ɑS expression in neurons. Our results identify convergent mechanisms across synucleinopathies, some shared with AD.

Secondary glaucoma: Toward interventions based on molecular underpinnings.

Glaucoma is a heterogeneous group of progressive diseases that leads to irreversible blindness. Secondary glaucoma refers to glaucoma caused by a known underlying condition. Pseudoexfoliation and pigment dispersion syndromes are common causes of secondary glaucoma. Their respective deposits may obstruct the trabecular meshwork, leading to aqueous humor outflow resistance, ocular hypertension, and optic neuropathy. There are no disease-specific interventions available for either. Pseudoexfoliation syndrome is characterized by fibrillar deposits (pseudoexfoliative material) on anterior segment structures. Over a decade of multiomics analyses taken together with the current knowledge on pseudoexfoliative glaucoma warrant a re-think of mechanistic possibilities. We propose that the presence of nucleation centers (e.g., vitamin D binding protein), crosslinking enzymes (e.g., transglutaminase 2), aberrant extracellular matrix, flawed endocytosis, and abnormal aqueous-blood barrier contribute to the formation of proteolytically resistant pseudoexfoliative material. Pigment dispersion syndrome is characterized by abnormal iridolenticular contact that disrupts iris pigment epithelium and liberates melanin granules. Iris melanogenesis is aberrant in this condition. Cytotoxic melanogenesis intermediates leak out of melanosomes and cause iris melanocyte and pigment epithelium cell death. Targeting melanogenesis can likely decrease the risk of pigmentary glaucoma. Skin and melanoma research provides insights into potential therapeutics. We propose that specific prostanoid agonists and fenofibrates may reduce melanogenesis by inhibiting cholesterol internalization and de novo synthesis. Additionally, melatonin is a potent melanogenesis suppressor, antioxidant, and hypotensive agent, rendering it a valuable agent for pigmentary glaucoma. In pseudoexfoliative glaucoma, where environmental insults drive pseudoexfoliative material formation, melatonin's antioxidant and hypotensive properties may offer adjunct therapeutic benefits. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology.

Multilayered mechanisms ensure that short chromosomes recombine in meiosis.

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes3-7 and are nearly universal in eukaryotes8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes.

Proteome-Scale Mapping of Perturbed Proteostasis in Living Cells.

Proteinopathies are degenerative diseases in which specific proteins adopt deleterious conformations, leading to the dysfunction and demise of distinct cell types. They comprise some of the most significant diseases of aging-from Alzheimer's disease to Parkinson's disease to type 2 diabetes-for which not a single disease-modifying or preventative strategy exists. Here, we survey approaches in tractable cellular and organismal models that bring us toward a more complete understanding of the molecular consequences of protein misfolding. These include proteome-scale profiling of genetic modifiers, as well as transcriptional and proteome changes. We describe assays that can capture protein interactomes in situ and distinct protein conformational states. A picture of cellular drivers and responders to proteotoxicity emerges from this work, distinguishing general alterations of proteostasis from cellular events that are deeply tied to the intrinsic function of the misfolding protein. These distinctions have consequences for the understanding and treatment of proteinopathies.

Necl-4/Cadm4 recruits Par-3 to the Schwann cell adaxonal membrane.

Interactions between axons and Schwann cells are essential for the acquisition of Schwann cell radial and longitudinal polarity and myelin sheath assembly. In the internode, the largest of these longitudinal domains, axon-Schwann cell interactions are mediated by the Nectin-like (Necl) cell adhesion proteins, also known as SynCAMs or Cadms. In particular, Necl-1/Cadm3 expressed on the axon surface binds to Necl-4/Cadm4 expressed along the adaxonal membrane of myelinating Schwann cells. Necl-4 promotes myelination in vitro and is required for the timely onset of myelination and the fidelity of the organization of the myelin sheath and the internode in vivo. A key question is the identity of the downstream effectors of Necl-4 that mediate its effects. The cytoplasmic terminal region (CTR) of Necl-4 contains a PDZ-domain binding motif. Accordingly, we used the CTR of Necl-4 in an unbiased proteomic screen of PDZ-domain proteins. We identify Par-3, a multi-PDZ domain containing protein of the Par-aPKC polarity complex previously implicated in myelination, as an interacting protein. Necl-4 and Par-3 are colocalized along the inner Schwann cell membrane and coprecipitate from Schwann cell lysates. The CTR of Necl-4 binds to the first PDZ domain of Par-3 thereby recruiting Par-3 to sites of Necl-4/Necl-1 interaction. Knockdown of Necl-4 perturbs Par-3 localization to the inner membrane of Schwann cells in myelinating co-cultures. These findings implicate interactions of Necl-1/Necl-4 in the recruitment of Par-3 to the Schwann cell adaxonal membrane and the establishment of Schwann cell radial polarity.

The mutational decay of male-male and hermaphrodite-hermaphrodite competitive fitness in the androdioecious nematode C. elegans.

Androdioecious Caenorhabditis have a high frequency of self-compatible hermaphrodites and a low frequency of males. The effects of mutations on male fitness are of interest for two reasons. First, when males are rare, selection on male-specific mutations is less efficient than in hermaphrodites. Second, males may present a larger mutational target than hermaphrodites because of the different ways in which fitness accrues in the two sexes. We report the first estimates of male-specific mutational effects in an androdioecious organism. The rate of male-specific inviable or sterile mutations is ⩽5 × 10-4/generation, below the rate at which males would be lost solely due to those kinds of mutations. The rate of mutational decay of male competitive fitness is ~ 0.17%/generation; that of hermaphrodite competitive fitness is ~ 0.11%/generation. The point estimate of ~ 1.5X faster rate of mutational decay of male fitness is nearly identical to the same ratio in Drosophila. Estimates of mutational variance (VM) for male mating success and competitive fitness are not significantly different from zero, whereas VM for hermaphrodite competitive fitness is similar to that of non-competitive fitness. Two independent estimates of the average selection coefficient against mutations affecting hermaphrodite competitive fitness agree to within two-fold, 0.33-0.5%.

Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast.

Meiosis is a specialized form of cell division resulting in reproductive cells with a reduced, usually haploid, genome complement. A key step after premeiotic DNA replication is the occurrence of homologous recombination at multiple places throughout the genome, initiated with the formation of DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like protein Spo11. DSBs are distributed non-randomly in genomes, and understanding the mechanisms that shape this distribution is important for understanding how meiotic recombination influences heredity and genome evolution. Several methods exist for mapping where Spo11 acts. Of these, sequencing of Spo11-associated oligonucleotides (Spo11 oligos) is the most precise, specifying the locations of DNA breaks to the base pair. In this chapter we detail the steps involved in Spo11-oligo mapping in the SK1 strain of budding yeast Saccharomyces cerevisiae, from harvesting cells of highly synchronous meiotic cultures, through preparation of sequencing libraries, to the mapping pipeline used for processing the data.

The kinetochore prevents centromere-proximal crossover recombination during meiosis.

During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes.

Nonparadoxical evolutionary stability of the recombination initiation landscape in yeast.

The nonrandom distribution of meiotic recombination shapes heredity and genetic diversification. Theoretically, hotspots--favored sites of recombination initiation--either evolve rapidly toward extinction or are conserved, especially if they are chromosomal features under selective constraint, such as promoters. We tested these theories by comparing genome-wide recombination initiation maps from widely divergent Saccharomyces species. We find that hotspots frequently overlap with promoters in the species tested, and consequently, hotspot positions are well conserved. Remarkably, the relative strength of individual hotspots is also highly conserved, as are larger-scale features of the distribution of recombination initiation. This stability, not predicted by prior models, suggests that the particular shape of the yeast recombination landscape is adaptive and helps in understanding evolutionary dynamics of recombination in other species.

Maternal rat serum concentrations of dimethadione do not explain intra-litter differences in the incidence of dimethadione-induced birth defects, including novel findings in foetal lung.

To investigate mechanisms of chemical-induced congenital heart defects (CHD) we have developed a rat model using dimethadione (DMO), the N-demethylated metabolite of the anticonvulsant, trimethadione (TMD). Dosing pregnant rats with 300mg/kg DMO every 12h from the evening of gestational day (GD) 8 until the morning of GD 11 (six total doses) produces a mean 74% incidence of CHD with inter litter variability ranging from 40 to 100%. The goal of this study was to determine if the variability in maternal serum concentrations of DMO on GD 14, a surrogate marker for total exposure, was related to the inter-litter differences in teratogenic outcomes. To test this hypothesis, pregnant rats were dosed as described above and serum levels of DMO assessed on GD 14. On GD 21, foetuses were collected by caesarean section, assessed for a number endpoints and the outcomes were correlated with the GD 14 serum concentrations of DMO. DMO exposure was associated with decreased foetal body weight, increased incidence of sternal defects and CHD, but these endpoints were not meaningfully correlated with maternal concentrations of DMO. Novel findings were decreased viability as measured one-hour following caesarean section, and delayed alveolar maturation. The major conclusions from these studies were first, that serum DMO concentrations on GD 14 did not predict teratogenicity, and second, delayed lung development may contribute to the decreased survival of foetuses at the time of caesarean section.

Mechanism and regulation of meiotic recombination initiation.

Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence.

Measuring life space in older adults with mild-to-moderate Alzheimer's disease using mobile phone GPS.

BACKGROUND: As an indicator of physical and cognitive functioning in community-dwelling older adults, there is increasing interest in measuring life space, defined as the geographical area a person covers in daily life. Typically measured through questionnaires, life space can be challenging to assess in amnestic dementia associated with Alzheimer's disease (AD). While global positioning system (GPS) technology has been suggested as a potential solution, there remains a lack of data validating GPS-based methods to measure life space in cognitively impaired populations. OBJECTIVE: The purpose of the study was to evaluate the construct validity of a GPS system to provide quantitative measurements of global movement for individuals with mild-to-moderate AD. METHODS: Nineteen community-dwelling older adults with mild-to-moderate AD (Mini-Mental State Examination score 14-28, age 70.7 ± 2.2 years) and 33 controls (CTL; age 74.0 ± 1.2 years) wore a GPS-enabled mobile phone during the day for 3 days. Measures of geographical territory (area, perimeter, mean distance from home, and time away from home) were calculated from the GPS log. Following a log-transformation to produce symmetrical distributions, group differences were tested using two-sample t tests. Construct validity of the GPS measures was tested by examining the correlation between the GPS measures and indicators of physical function [steps/day, gait velocity, and Disability Assessment for Dementia (DAD)] and affective state (Apathy Evaluation Scale and Geriatric Depression Scale). Multivariate regression was performed to evaluate the relative strength of significantly correlated factors. RESULTS: GPS-derived area (p < 0.01), perimeter (p < 0.01), and mean distance from home (p < 0.05) were smaller in the AD group compared to CTL. The correlation analysis found significant associations of the GPS measures area and perimeter with all measures of physical function (steps/day, DAD, and gait velocity; p < 0.01), symptoms of apathy (p < 0.01), and depression (p < 0.05). Multivariate regression analysis indicated that gait velocity and dependence were the strongest variables associated with GPS measures. CONCLUSION: This study demonstrated that GPS-derived area and perimeter: (1) distinguished mild-to-moderate AD patients from CTL and (2) were strongly correlated with physical function and affective state. These findings confirm the ability of GPS technology to assess life space behaviour and may be particularly valuable to continuously monitor functional decline associated with neurodegenerative disease, such as AD.

The 4.1B cytoskeletal protein regulates the domain organization and sheath thickness of myelinated axons.

Myelinated axons are organized into specialized domains critical to their function in saltatory conduction, i.e., nodes, paranodes, juxtaparanodes, and internodes. Here, we describe the distribution and role of the 4.1B protein in this organization. 4.1B is expressed by neurons, and at lower levels by Schwann cells, which also robustly express 4.1G. Immunofluorescence and immuno-EM demonstrates 4.1B is expressed subjacent to the axon membrane in all domains except the nodes. Mice deficient in 4.1B have preserved paranodes, based on marker staining and EM in contrast to the juxtaparanodes, which are substantially affected in both the PNS and CNS. The juxtaparanodal defect is evident in developing and adult nerves and is neuron-autonomous based on myelinating cocultures in which wt Schwann cells were grown with 4.1B-deficient neurons. Despite the juxtaparanodal defect, nerve conduction velocity is unaffected. Preservation of paranodal markers in 4.1B deficient mice is associated with, but not dependent on an increase of 4.1R at the axonal paranodes. Loss of 4.1B in the axon is also associated with reduced levels of the internodal proteins, Necl-1 and Necl-2, and of alpha-2 spectrin. Mutant nerves are modestly hypermyelinated and have increased numbers of Schmidt-Lanterman incisures, increased expression of 4.1G, and express a residual, truncated isoform of 4.1B. These results demonstrate that 4.1B is a key cytoskeletal scaffold for axonal adhesion molecules expressed in the juxtaparanodal and internodal domains that unexpectedly regulates myelin sheath thickness.

Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains.

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

Exploiting spore-autonomous fluorescent protein expression to quantify meiotic chromosome behaviors in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae has proven to be a rich source of information about the mechanisms and regulation of homologous recombination during meiosis. A common technique for studying this process involves microdissecting the four products (ascospores) of a single meiosis and analyzing the configuration of genetic markers in the spores that are viable. Although this type of analysis is powerful, it can be laborious and time-consuming to characterize the large numbers of meioses needed to generate statistically robust data sets. Moreover, the reliance on viable (euploid) spores has the potential to introduce selection bias, especially when analyzing mutants with elevated frequencies of meiotic chromosome missegregation. To overcome these limitations, we developed a versatile, portable set of reporter constructs that drive fluorescent protein expression specifically in only those spores that inherit the reporter. These spore-autonomous fluorescence constructs allow direct visualization of inheritance patterns in intact tetrads, eliminating the need for microdissection and permitting meiotic segregation patterns to be ascertained even in aneuploid spores. As proof of principle, we demonstrate how different arrangements of reporters can be used to quantify crossover frequency, crossover interference, gene conversion, crossover/noncrossover ratios, and chromosome missegregation.

Analyses of the yeast Rad51 recombinase A265V mutant reveal different in vivo roles of Swi2-like factors.

The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair.

Apathy symptom profile and behavioral associations in frontotemporal dementia vs dementia of Alzheimer type.

BACKGROUND: Apathy is a common and significant problem in patients with dementia, regardless of its cause. Observations about frontosubcortical circuit syndromes indicate that apathy may have affective, behavioral, or cognitive manifestations. OBJECTIVES: To explore whether the apathy manifested in frontotemporal dementia (FTD), with its predominantly anterior brain neuropathologic features, differs from the apathy in dementia of Alzheimer type (DAT), with its predominantly hippocampal- and temporoparietal-based neuropathologic features, and to determine whether other behavioral disturbances reported in frontosubcortical circuit syndromes correlate with apathy. DESIGN: Analyses included individual items within Neuropsychiatric Inventory subscale items. Items of the apathy/indifference subscale were designated by consensus as affective (lacking in emotions), behavioral (inactive, chores abandoned), or cognitive (no interest in the activities of others). Proportions of correlated nonapathy Neuropsychiatric Inventory items were calculated. SETTING: Several neurology specialty clinics contributed to our data set. PARTICIPANTS: A total of 92 participants with FTD and 457 with DAT. MAIN OUTCOME MEASURES: The Neuropsychiatric Inventory was analyzed. RESULTS: Apathy was more prevalent in patients with FTD than in those with DAT, but when present, the specific apathy symptoms associated with both types of dementia were rarely restricted to 1 of the 3 domains of apathy. Dysphoria concurrent with apathy was unique to the DAT group and negatively correlated in the FTD group. Participants with affective apathy more frequently copresented with an orbital frontosubcortical syndrome in FTD (impulsivity and compulsions). Affective apathy also copresented with uncooperative agitation, anger, and physical agitation in both types of dementia. CONCLUSIONS: Apathy is common in patients with FTD and DAT, although it is more common in those with FTD. When present, it usually involves changes in affect, behavior, and cognition. It is associated with behaviors that have previously been shown to affect patient safety, independence, and quality of life.

Evidence of normal hearing laterality in familial schizophrenic patients and their relatives.

Dichotic listening (DL) has been used as a tool to investigate possible left cerebral dysfunction in schizophrenia. However, the wide range of DL tests (e.g., words, emotions, sentences) as well as patient groups ("heterogeneity") has introduced several confounders. Assessing relatives of patients with schizophrenia may overcome some of these problems, and may be more useful in determining if loss of functional cerebral laterality in schizophrenia is a state or a trait phenomenon. The fused consonant-vowel DL test was administered to 114 subjects: 20 individuals with familial schizophrenia, 42 of their healthy relatives, and 52 healthy volunteers. We did this to investigate whether the normal language processing asymmetry-a right ear advantage (REA)-is present, and whether it could serve as a marker for genetic liability. General performance accuracy level was lower in schizophrenia patients and their relatives but the expected REA was present in all groups. Adjusting for age, accuracy, and obligate status made no difference. In conclusion, familial schizophrenic patients and their relatives have normal REA and hearing laterality on the fused DL test.

Nectin-like proteins mediate axon Schwann cell interactions along the internode and are essential for myelination.

Axon-glial interactions are critical for the induction of myelination and the domain organization of myelinated fibers. Although molecular complexes that mediate these interactions in the nodal region are known, their counterparts along the internode are poorly defined. We report that neurons and Schwann cells express distinct sets of nectin-like (Necl) proteins: axons highly express Necl-1 and -2, whereas Schwann cells express Necl-4 and lower amounts of Necl-2. These proteins are strikingly localized to the internode, where Necl-1 and -2 on the axon are directly apposed by Necl-4 on the Schwann cell; all three proteins are also enriched at Schmidt-Lanterman incisures. Binding experiments demonstrate that the Necl proteins preferentially mediate heterophilic rather than homophilic interactions. In particular, Necl-1 on axons binds specifically to Necl-4 on Schwann cells. Knockdown of Necl-4 by short hairpin RNA inhibits Schwann cell differentiation and subsequent myelination in cocultures. These results demonstrate a key role for Necl-4 in initiating peripheral nervous system myelination and implicate the Necl proteins as mediators of axo-glial interactions along the internode.

Nodes of Ranvier and axon initial segments are ankyrin G-dependent domains that assemble by distinct mechanisms.

Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and betaIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.