Location of the axon initial segment assembly can be predicted from neuronal shape.

The axon initial segment (AIS) is located at the proximal axon demarcating the boundary between axonal and somatodendritic compartments. The AIS facilitates the generation of action potentials and maintenance of neuronal polarity. In this study, we show that the location of AIS assembly, as marked by Ankyrin G, corresponds to the nodal plane of the lowest-order harmonic of the Laplace-Beltrami operator solved over the neuronal shape. This correlation establishes a coupling between location of AIS assembly and neuronal cell morphology. We validate this correlation for neurons with atypical morphology and neurons containing multiple AnkG clusters on distinct neurites, where the nodal plane selects the appropriate axon showing enriched Tau. Based on our findings, we propose that Turing patterning systems are candidates for dynamically governing AIS location. Overall, this study highlights the importance of neuronal cell morphology in determining the precise localization of the AIS within the proximal axon.

Tau target identification reveals NSF-dependent effects on AMPA receptor trafficking and memory formation.

Microtubule-associated protein tau is a central factor in Alzheimer's disease and other tauopathies. However, the physiological functions of tau are unclear. Here, we used proximity-labelling proteomics to chart tau interactomes in primary neurons and mouse brains in vivo. Tau interactors map onto pathways of cytoskeletal, synaptic vesicle and postsynaptic receptor regulation and show significant enrichment for Parkinson's, Alzheimer's and prion disease. We find that tau interacts with and dose-dependently reduces the activity of N-ethylmaleimide sensitive fusion protein (NSF), a vesicular ATPase essential for AMPA-type glutamate receptor (AMPAR) trafficking. Tau-deficient (tau-/- ) neurons showed mislocalised expression of NSF and enhanced synaptic AMPAR surface levels, reversible through the expression of human tau or inhibition of NSF. Consequently, enhanced AMPAR-mediated associative and object recognition memory in tau-/- mice is suppressed by both hippocampal tau and infusion with an NSF-inhibiting peptide. Pathologic mutant tau from mouse models or Alzheimer's disease significantly enhances NSF inhibition. Our results map neuronal tau interactomes and delineate a functional link of tau with NSF in plasticity-associated AMPAR-trafficking and memory.

The Nature of Diamino Linker and Halogen Bonding Define Selectivity of Pyrrolopyrimidine-Based LIMK1 Inhibitors.

The LIM-domain kinase (LIMK) family consists of two isoforms, LIMK1 and LIMK2, which are highly homologous, making selective inhibitor development challenging. LIMK regulates dynamics of the actin cytoskeleton, thereby impacting many cellular functions including cell morphology and motility. Here, we designed and synthesised analogues of a known pyrrolopyrimidine LIMK inhibitor with moderate selectivity for LIMK1 over LIMK2 to gain insights into which features contribute to both activity and selectivity. We incorporated a different stereochemistry around a cyclohexyl central moiety to achieve better selectivity for different LIMK isoforms. Inhibitory activity was assessed by kinase assays, and biological effects in cells were determined using an in vitro wound closure assay. Interestingly, a slight change in stereochemistry alters LIMK isoform selectivity. Finally, a docking study was performed to predict how the new compounds interact with the target.

Overexpression of Tropomyosin Isoform Tpm3.1 Does Not Alter Synaptic Function in Hippocampal Neurons.

Tropomyosin (Tpm) has been regarded as the master regulator of actin dynamics. Tpms regulate the binding of the various proteins involved in restructuring actin. The actin cytoskeleton is the predominant cytoskeletal structure in dendritic spines. Its regulation is critical for spine formation and long-term activity-dependent changes in synaptic strength. The Tpm isoform Tpm3.1 is enriched in dendritic spines, but its role in regulating the synapse structure and function is not known. To determine the role of Tpm3.1, we studied the synapse structure and function of cultured hippocampal neurons from transgenic mice overexpressing Tpm3.1. We recorded hippocampal field excitatory postsynaptic potentials (fEPSPs) from brain slices to examine if Tpm3.1 overexpression alters long-term synaptic plasticity. Tpm3.1-overexpressing cultured neurons did not show a significantly altered dendritic spine morphology or synaptic activity. Similarly, we did not observe altered synaptic transmission or plasticity in brain slices. Furthermore, expression of Tpm3.1 at the postsynaptic compartment does not increase the local F-actin levels. The results suggest that although Tpm3.1 localises to dendritic spines in cultured hippocampal neurons, it does not have any apparent impact on dendritic spine morphology or function. This is contrary to the functional role of Tpm3.1 previously observed at the tip of growing neurites, where it increases the F-actin levels and impacts growth cone dynamics.

Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons-New Insights into the Role of the C-Terminal Region of Tpm3.1.

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

Rapid initiation of cell cycle reentry processes protects neurons from amyloid-ß toxicity.

Neurons are postmitotic cells. Reactivation of the cell cycle by neurons has been reported in Alzheimer's disease (AD) brains and models. This gave rise to the hypothesis that reentering the cell cycle renders neurons vulnerable and thus contributes to AD pathogenesis. Here, we use the fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology to monitor the cell cycle in live neurons. We found transient, self-limited cell cycle reentry activity in naive neurons, suggesting that their postmitotic state is a dynamic process. Furthermore, we observed a diverse response to oligomeric amyloid-ß (oAß) challenge; neurons without cell cycle reentry activity would undergo cell death without activating the FUCCI reporter, while neurons undergoing cell cycle reentry activity at the time of the oAß challenge could maintain and increase FUCCI reporter signal and evade cell death. Accordingly, we observed marked neuronal FUCCI positivity in the brains of human mutant Aß precursor protein transgenic (APP23) mice together with increased neuronal expression of the endogenous cell cycle control protein geminin in the brains of 3-mo-old APP23 mice and human AD brains. Taken together, our data challenge the current view on cell cycle in neurons and AD, suggesting that pathways active during early cell cycle reentry in neurons protect from Aß toxicity.

CYLD is a causative gene for frontotemporal dementia - amyotrophic lateral sclerosis.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD's interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

Functional characterisation of filamentous actin probe expression in neuronal cells.

Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.