Synapses display a stereotyped ultrastructural organization, commonly containing a single electron-dense presynaptic density surrounded by a cluster of synaptic vesicles. The mechanism controlling subsynaptic proportion is not understood. Loss of function in the C. elegans rpm-1 gene, a putative RING finger/E3 ubiquitin ligase, causes disorganized presynaptic cytoarchitecture. RPM-1 is localized to the presynaptic periactive zone. We report that RPM-1 negatively regulates a p38 MAP kinase pathway composed of the dual leucine zipper-bearing MAPKKK DLK-1, the MAPKK MKK-4, and the p38 MAP kinase PMK-3. Inactivation of this pathway suppresses rpm-1 loss of function phenotypes, whereas overexpression or constitutive activation of this pathway causes synaptic defects resembling rpm-1(lf) mutants. DLK-1, like RPM-1, is localized to the periactive zone. DLK-1 protein levels are elevated in rpm-1 mutants. The RPM-1 RING finger can stimulate ubiquitination of DLK-1. Our data reveal a presynaptic role of a previously unknown p38 MAP kinase cascade.

The molecular mechanisms regulating fate divergence of closely related, but distinct, layer 6 corticothalamic and layer 5 subcerebral projection neurons are largely unknown. We present evidence for central transcriptional mechanisms that regulate fate specification of corticothalamic (layer 6) and subcerebral (layer 5) projection neurons. We found that TBR1 promotes the identity of corticothalamic neurons and represses subcerebral fates through reducing expression of Fezf2 and CTIP2. These conclusions are based on: (1) In Tbr1−/− mice, the number of cells expressing layer 6 markers was reduced, and the number of cells expressing layer 5 markers was increased. Early-born (birthdated on E11.5) neurons ectopically expressed subcerebral neuronal markers, and extended their axons into subcerebral targets; (2) Ectopic Tbr1 expression in layer 5 neurons prevented them from extending axons into the brain stem and the spinal cord; (3) ChIP analysis using TBR1 antibodies showed that TBR1 bound to a conserved region in the Fezf2 gene; (4) Analysis of Fezf2 mutants and Tbr1−/−; Fezf2−/− compound mutants provided evidence that Fezf2 blocks corticothalamic fate in layer 5 by reducing Tbr1 expression in subcerebral neurons. All neocortical regions appear to use this core transcriptional program to specify corticothalamic (layer 6) and subcerebral (layer 5) projection neurons.

During synapse formation, specialized subcellular structures develop at synaptic junctions in a tightly regulated fashion. Cross-signalling initiated by ephrins, Wnts and transforming growth factor-beta family members between presynaptic and postsynaptic termini are proposed to govern synapse formation. It is not well understood how multiple signals are integrated and regulated by developing synaptic termini to control synaptic differentiation. Here we report the identification of FSN-1, a novel F-box protein that is required in presynaptic neurons for the restriction and/or maturation of synapses in Caenorhabditis elegans. Many F-box proteins are target recognition subunits of SCF (Skp, Cullin, F-box) ubiquitin-ligase complexes. fsn-1 functions in the same pathway as rpm-1, a gene encoding a large protein with RING finger domains. FSN-1 physically associates with RPM-1 and the C. elegans homologues of SKP1 and Cullin to form a new type of SCF complex at presynaptic periactive zones. We provide evidence that T10H9.2, which encodes the C. elegans receptor tyrosine kinase ALK (anaplastic lymphoma kinase), may be a target or a downstream effector through which FSN-1 stabilizes synapse formation. This neuron-specific, SCF-like complex therefore provides a localized signal to attenuate presynaptic differentiation.

Racemates often have lower solubility than enantiopure compounds, and mixing of enantiomers can enhance aggregation propensity of peptides. Amyloid β (Aβ) 42 is an aggregation-prone peptide, believed to play a key role in Alzheimer’s Disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as particularly neurotoxic. We hypothesized that addition of mirror image (D-) Aβ42 should reduce the concentration of toxic oligomers formed by natural (L-) Aβ42. We synthesized L- and D-Aβ42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogs of the enantiomers showed their co-localization in racemic fibrils. Reflecting enhanced fibril formation propensity, racemic Aβ42 was less prone to form soluble oligomers. This resulted in protection of cells from toxicity of L-Aβ42 at concentrations ranging up to 50 µM. In summary, mixing of Aβ42 enantiomers induces accelerated formation of non-toxic fibrils.

Amyloid-β (Aβ) is an intrinsically disordered peptide thought to play an important role in Alzheimer's disease (AD). It has been the target of most AD therapeutic efforts, which have repeatedly failed in clinical trials. A more predominant peptidic fragment, formed through alternative processing of the amyloid precursor protein, is the p3 peptide. p3 has received little attention, which is possibly due to the prevailing view in the AD field that it is "non-amyloidogenic." By probing the self-assembly of this peptide, we found that p3 aggregates to form oligomers and fibrils and, when compared with Aβ, displays enhanced aggregation rates. Our findings highlight the solubilizing effect of the Nterminus of Aβ and the favorable formation of structures formed through C-terminal hydrophobic peptide interfaces. Based on our findings, we suggest a reevaluation of the current therapeutic approaches targeting only the β-secretase pathway of AD, given that the αsecretase pathway is also amyloidogenic.

Racemic mixtures frequently display properties that are different from those associated with their enantiopure counterparts, and are often characterized by higher propensity to form aggregates. Our previous research established that mixing of the enantiomers of Alzheimer amyloid β (Aβ) 42 peptides is an effective strategy to induce an oligomer‐to‐fibril conversion, which puts Aβ42 into a substantially less toxic state. Here, new insights into this chiral inactivation effect are presented. In addition to the commonly used Thioflavin T fibril formation assays, the use of the less aggregation‐prone Aβ40 system allowed us to monitor peptide aggregation by NMR. Whereas enantiopure peptide was well soluble under the chosen experimental conditions and showed no sign of precipitation, addition of one equivalent of the mirror‐image peptide triggered an instant and rapid aggregation, observable through the attenuation of the NMR signal. The racemic Aβ40 fibrils were found by transmission electron microscopy to be distinct in morphology, exhibiting a ~2‐fold narrowing as compared with their enantiopure counterparts.

Caenorhabditis elegans RPM-1 is a member of a conserved protein family, the PHR proteins, that includes human Pam, mouse Phr1, zebrafish Esrom, and Drosophila Highwire. PHR proteins play important roles in the development of the nervous system. In particular, mutations in rpm-1 cause a disruption of synaptic architecture, affecting the distribution of synaptic vesicles and the number of presynaptic densities. Using antibodies against RPM-1, we determined the localization of the endogenous RPM-1 protein in wild-type and in several mutants that affect synaptic development. Our analyses show that, in mature neurons, RPM-1 resides in a distinct region that is close to, but does not overlap with, the synaptic exo-and endocytosis domains. The localization of RPM-1 occurs independently of several proteins that function in the transport or assembly of synapse components, and its abundance is partially dependent on its binding partner the F-box protein FSN-1. RPM-1 has been shown to target the MAPKKK DLK-1 for degradation. We show that activated DLK-1 may be preferentially targeted for degradation. Furthermore, using transgene analysis, we identified a critical role of the conserved PHR domain of RPM-1 in its subcellular localization.

Amyloid β is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42‐amino‐acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloid β and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of ≈5:1. This suggests that the process is predominantly receptor‐mediated, with likely minor contributions of non‐stereoselective mechanisms.