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1. Inhibition of AKT Signaling Alters βIV Spectrin Distribution at the AIS and Increases Neuronal Excitability

   https://doi.org/10.3389/fnmol.2021.643860  

   Di Re, Jessica; Hsu, Wei-Chun J.; Kayasandik, Cihan B.; Fularczyk, Nickolas; James, T. F.; Nenov, Miroslav N.; Negi, Pooran; Marosi, Mate; Scala, Federico; Prasad, Saurabh; et al (June 2021, Frontiers in Molecular Neuroscience)

   null (Ed.)

   The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, βIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing βIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on βIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in βIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders. 

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2. Homeostatic regulation of extracellular signal-regulated kinase 1/2 activity and axonal Kv7.3 expression by prolonged blockade of hippocampal neuronal activity

   https://doi.org/10.3389/fncel.2022.838419  

   Baculis, Brian C.; Kesavan, Harish; Weiss, Amanda C.; Kim, Edward H.; Tracy, Gregory C.; Ouyang, Wenhao; Tsai, Nien-Pei; Chung, Hee Jung (July 2022, Frontiers in Cellular Neuroscience)

   Homeostatic plasticity encompasses the mechanisms by which neurons stabilize their synaptic strength and excitability in response to prolonged and destabilizing changes in their network activity. Prolonged activity blockade leads to homeostatic scaling of action potential (AP) firing rate in hippocampal neurons in part by decreased activity of N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes includingKCNQ3which encodes K_v7.3. Neuronal K_v7 channels are mostly heterotetramers of K_v7.2 and K_v7.3 subunits and are highly enriched at the axon initial segment (AIS) where their current potently inhibits repetitive and burst firing of APs. However, whether a decrease in K_v7.3 expression occurs at the AIS during homeostatic scaling of intrinsic excitability and what signaling pathway reducesKCNQ3transcript upon prolonged activity blockade remain unknown. Here, we report that prolonged activity blockade in cultured hippocampal neurons reduces the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) followed by a decrease in the activation of brain-derived neurotrophic factor (BDNF) receptor, Tropomyosin receptor kinase B (TrkB). Furthermore, both prolonged activity blockade and prolonged pharmacological inhibition of ERK1/2 decreaseKCNQ3andBDNFtranscripts as well as the density of K_v7.3 and ankyrin-G at the AIS. Collectively, our findings suggest that a reduction in the ERK1/2 activity and subsequent transcriptional down-regulation may serve as a potential signaling pathway that links prolonged activity blockade to homeostatic control of BDNF-TrkB signaling and K_v7.3 density at the AIS during homeostatic scaling of AP firing rate. 

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3. Differential Control of Small-conductance Calcium-activated Potassium Channel Diffusion by Actin in Different Neuronal Subcompartments

   https://doi.org/10.1093/function/zqad018  

   Gu, Shiju; Tzingounis, Anastasios V; Lykotrafitis, George (January 2023, Function)

   Abstract Small-conductance calcium-activated potassium (SK) channels show a ubiquitous distribution on neurons, in both somatodendritic and axonal regions. SK channels are associated with neuronal activity regulating action potential frequency, dendritic excitability, and synaptic plasticity. Although the physiology of SK channels and the mechanisms that control their surface expression levels have been investigated extensively, little is known about what controls SK channel diffusion in the neuronal plasma membrane. This aspect is important, as the diffusion of SK channels at the surface may control their localization and proximity to calcium channels, hence increasing the likelihood of SK channel activation by calcium. In this study, we successfully investigated the diffusion of SK channels labeled with quantum dots on human embryonic kidney cells and dissociated hippocampal neurons by combining a single-particle tracking method with total internal reflection fluorescence microscopy. We observed that actin filaments interfere with SK mobility, decreasing their diffusion coefficient. We also found that during neuronal maturation, SK channel diffusion was gradually inhibited in somatodendritic compartments. Importantly, we observed that axon barriers formed at approximately days in vitro 6 and restricted the diffusion of SK channels on the axon initial segment (AIS). However, after neuron maturation, SK channels on the AIS were strongly immobilized, even after disruption of the actin network, suggesting that crowding may cause this effect. Altogether, our work provides insight into how SK channels diffuse on the neuronal plasma membrane and how actin and membrane crowding impacts SK channel diffusion. 

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4. The Amyloid Precursor Protein Modulates the Position and Length of the Axon Initial Segment

   https://doi.org/10.1523/jneurosci.0172-22.2023  

   Ma, Fulin; Akolkar, Himanshu; Xu, Jianquan; Liu, Yang; Popova, Dina; Xie, Jiaan; Youssef, Mark M.; Benosman, Ryad; Hart, Ronald P.; Herrup, Karl (March 2023, The Journal of Neuroscience)

   The amyloid precursor protein (APP) is linked to the genetics and pathogenesis of Alzheimer's disease (AD). It is the parent protein of the β-amyloid (Aβ) peptide, the main constituent of the amyloid plaques found in an AD brain. The pathways from APP to Aβ are intensively studied, yet the normal functions of APP itself have generated less interest. We report here that glutamate stimulation of neuronal activity leads to a rapid increase inAppgene expression. In mouse and human neurons, elevated APP protein changes the structure of the axon initial segment (AIS) where action potentials are initiated. The AIS is shortened in length and shifts away from the cell body. The GCaMP8f Ca²⁺reporter confirms the predicted decrease in neuronal activity. NMDA antagonists or knockdown ofAppblock the glutamate effects. The actions of APP on the AIS are cell-autonomous; exogenous Aβ, either fibrillar or oligomeric, has no effect. In culture, APP_Swe(a familial AD mutation) induces larger AIS changes than wild type APP. Ankyrin G and βIV-spectrin, scaffolding proteins of the AIS, both physically associate with APP, more so in AD brains. Finally, in humans with sporadic AD or in the R1.40 AD mouse model, both females and males, neurons have elevated levels of APP protein that invade the AIS.In vivoasin vitro, this increased APP is associated with a significant shortening of the AIS. The findings outline a new role for the APP and encourage a reconsideration of its relationship to AD. SIGNIFICANCE STATEMENTWhile the amyloid precursor protein (APP) has long been associated with Alzheimer's disease (AD), the normal functions of the full-length Type I membrane protein have been largely unexplored. We report here that the levels of APP protein increase with neuronal activity.In vivoandin vitro, modest amounts of excess APP alter the properties of the axon initial segment. The β-amyloid peptide derived from APP is without effect. Consistent with the observed changes in the axon initial segment which would be expected to decrease action potential firing, we show that APP expression depresses neuronal activity. In mouse AD models and human sporadic AD, APP physically associates with the scaffolding proteins of the axon initial segment, suggesting a relationship with AD dementia. 

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5. Extracellular Tau Oligomers Damage the Axon Initial Segment by an Endogenous Tau‐Dependent Mechanism

   https://doi.org/10.1002/alz.078491  

   Best, Merci N; Lim, Yunu; Ferenc, Nina; Kim, Nayoung; Wang, Dora Bigler; Sharifi, Kamyar; Wasserman, Anna; McTavish, Sloane; Siller, Karsten; Jones, Marieke K; et al (December 2023, Alzheimer's & Dementia)

   Abstract BackgroundNeuronal polarity and synaptic connectivity are compromised in Alzheimer’s disease (AD) and other tauopathies. The axon initial segment (AIS) is a key structure for regulating polarity and functions of neurons. It occupies the first 20‐60 µm of the axon, comprises a diffusion barrier that segregates axon‐enriched from somatodendritic‐enriched molecules, and has a high concentration of voltage‐gated ion channels that generate action potentials. Extracellular amyloid‐β oligomers compromise AIS integrity. However, effects on the AIS of toxic tau species, including extracellular oligomers (xcTauOs) that spread tau pathology from neuron to neuron by a prion‐like process, whereas unknown. Therefore, we wanted to test the hypothesis that AIS structure is sensitive to xcTauOs. MethodPrimary cortical neurons derived from either wild type (WT), or tau knockout (KO) mice were exposed to xcTauOs or vehicle. Quantitative western blotting and immunofluorescence microscopy with an antibody against the AIS‐enriched protein TRIM46 was used to monitor effects on the AIS. The same methods were also used to compare TRIM46 and two other AIS proteins, ankyrin‐G and neurofascin‐186 in human hippocampal tissue obtained from AD and age‐matched non‐AD donors. ResultIn cultured WT, but not TKO neurons, xcTauOs cause a trend toward AIS shortening and reduce the concentration of the resident AIS protein, TRIM46, without affecting total TRIM46 levels. Lentiviral‐driven human tau expression in tau KO neurons rescues TRIM46 sensitivity to xcTauOs. In human AD hippocampus, AIS length and TRIM46 concentration within the AIS are reduced in neurons containing neurofibrillary tangles (NFTs), without affecting the overall protein levels of multiple resident AIS proteins. ConclusionThese collective findings demonstrate that in cultured neurons, xcTauOs cause partial AIS damage by a mechanism dependent on intracellular tau, thereby raising the possibility that AIS reduction in AD is caused by xcTauOs working in concert with endogenous neuronal tau. 

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