Use of Phosphatide Precursors to Promote Synaptogenesis

The actin-based dynamics of dendritic spines play a key role in synaptic plasticity, which underlies learning and memory. Although it is becoming increasingly clear that modulation of actin is critical for spine dynamics, the upstream molecular signals that regulate the formation and plasticity of spines are poorly understood. In non-neuronal cells, integrins are critical modulators of the actin cytoskeleton, but their function in the nervous system is not well characterized. Here we show that alpha5 integrin regulates spine morphogenesis and synapse formation in hippocampal neurons. Knockdown of alpha5 integrin expression using small interfering RNA decreased the number of dendritic protrusions, spines, and synapses. Expression of constitutively active or dominant negative alpha5 integrin also resulted in alterations in the number of dendritic protrusions, spines, and synapses. alpha5 integrin signaling regulates spine morphogenesis and synapse formation by a mechanism that is dependent on Src kinase, Rac, and the signaling adaptor GIT1. Alterations in the activity or localization of these molecules result in a significant decrease in the number of spines and synapses. Thus, our results point to a critical role for integrin signaling in regulating the formation of dendritic spines and synapses in hippocampal neurons.

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.

Alzheimer disease (AD) is characterized by neurodegeneration marked by loss of synapses and spines associated with hyperphosphorylation of tau protein. Accumulating amyloid β peptide (Aβ) in brain is linked to neurofibrillary tangles composed of hyperphosphorylated tau in AD. Here, we identify β2-adrenergic receptor (β2AR) that mediates Aβ-induced tau pathology. In the prefrontal cortex (PFC) of 1-year-old transgenic mice with human familial mutant genes of presenilin 1 and amyloid precursor protein (PS1/APP), the phosphorylation of tau at Ser-214 Ser-262 and Thr-181, and the protein kinases including JNK, GSK3α/β, and Ca(2+)/calmodulin-dependent protein kinase II is increased significantly. Deletion of the β2AR gene in PS1/APP mice greatly decreases the phosphorylation of these proteins. Further analysis reveals that in primary PFC neurons, Aβ signals through a β2AR-PKA-JNK pathway, which is responsible for most of the phosphorylation of tau at Ser-214 and Ser-262 and a significant portion of phosphorylation at Thr-181. Aβ also induces a β2AR-dependent arrestin-ERK1/2 activity that does not participate in phosphorylation of tau. However, inhibition of the activity of MEK, an upstream enzyme of ERK1/2, partially blocks Aβ-induced tau phosphorylation at Thr-181. The density of dendritic spines and synapses is decreased in the deep layer of the PFC of 1-year-old PS1/APP mice, and the mice exhibit impairment of learning and memory in a novel object recognition paradigm. Deletion of the β2AR gene ameliorates pathological effects in these senile PS1/APP mice. The study indicates that β2AR may represent a potential therapeutic target for preventing the development of AD.

Alzheimer's disease (AD), a multifactorial disease, is characterized by the accumulation of neurofibrillary tangles (NFTs) and amyloid beta (Aβ) plaques. AD is triggered via several factors like alteration in cytoskeletal proteins, a mutation in presenilin 1 (PSEN1), presenilin 2 (PSEN2), amyloid precursor protein (APP), and post-translational modifications (PTMs) in the cytoskeletal elements. Owing to the major structural and functional role of cytoskeletal elements, like the organization of axon initial segmentation, dendritic spines, synaptic regulation, and delivery of cargo at the synapse; modulation of these elements plays an important role in AD pathogenesis; like Tau is a microtubule-associated protein that stabilizes the microtubules, and it also causes inhibition of nucleo-cytoplasmic transportation by disrupting the integrity of nuclear pore complex. One of the major cytoskeletal elements, actin and its dynamics, regulate the dendritic spine structure and functions; impairments have been documented towards learning and memory defects. The second major constituent of these cytoskeletal elements, microtubules, are necessary for the delivery of the cargo, like ion channels and receptors at the synaptic membranes, whereas actin-binding protein, i.e., Cofilin's activation form rod-like structures, is involved in the formation of paired helical filaments (PHFs) observed in AD. Also, the glial cells rely on their cytoskeleton to maintain synaptic functionality. Thus, making cytoskeletal elements and their regulation in synaptic structure and function as an important aspect to be focused for better management and targeting AD pathology. This review advocates exploring phytochemicals and Ayurvedic plant extracts against AD by elucidating their neuroprotective mechanisms involving cytoskeletal modulation and enhancing synaptic plasticity. However, challenges include their limited bioavailability due to the poor solubility and the limited potential to cross the blood-brain barrier (BBB), emphasizing the need for targeted strategies to improve therapeuticefficacy.

Secretory sphingomyelinases are released in response to various types of stress stimuli, including inflammation [1]. Excessive activity and release of these enzymes can be pathological and accelerate age-related neurological changes, neuroinflammation, neurodegenerative disorders, and muscle dysfunction [1–3]. In contrast, sphingomyelinases were detected in areas close to exocytosis sites and dendritic spines, and their inhibition reduced synaptic transmission at both the presynaptic and postsynaptic levels in hippocampal synapses. Deficiency of acid sphingomyelinase results in neurodegenerative Niemann–Pick disease type A. Knockouts of neutral sphingomyelinases in model mice cause motor defects and disorders that resemble the Alzheimer’s disease phenotype [1, 2]. These findings suggest the existence of a mechanism regulating synaptic transmission, which is dependent on sphingomyelinases. In this study, we examined the impact of neutral sphingomyelinase on neurotransmission in mouse diaphragm muscles using microelectrode recordings of postsynaptic responses, exo-endocytic FM dyes, and fluorescent probes sensitive to membrane properties. The researchers discovered that adding sphingomyelinase at a low concentration (0.01 u/ml) for 15 minutes causes disruption of the lipid packaging of selectively synaptic membranes. This disruption leads to ceramide accumulation, a decrease in staining with Alexa Fluor 488 cholera toxin subunit B conjugate (a marker of GM1 ganglioside cluster), and a shift to the green part of the F2N12S fluorescence spectrum (an indicator of the expansion of the lipid-disordered phase). Increased fluorescence of 22-NBD-cholesterol, indicating an increase in membrane fluidity, and enhanced incorporation of exogenous ceramide into the outer monolayer were observed in the synaptic membranes. Increasing sphingomyelinase concentration to 0.1 u/ml intensifies changes in synaptic membranes and leads to corresponding alterations in membrane properties in muscle fiber plasmalemma. Subsequent experiments used a synapse-specific effect on membrane properties by applying sphingomyelinase at a low concentration for 15 minutes. The application of sphingomyelinase did not alter the amplitude and temporal parameters of miniature postsynaptic responses or the resting membrane potential. Additionally, sphingomyelinase (0.01 u/ml) did not change spontaneous secretion and evoked neurotransmitter release in response to single stimuli. However, the activity of sphingomyelinase led to a notable increase in the release of neurotransmitters and the rate of exocytotic unloading of FM dye from synaptic vesicles during nerve stimulation at 10, 20, and 70 Hz frequencies. The effect of sphingomyelinase on neurotransmission potentiation was irreversible and more significant at 10 Hz activity. Experiments involving intermittent nerve stimulation with short bursts of 60 stimuli each at 10 or 20 Hz frequencies, along with brief 0.5-second rest intervals, indicated that sphingomyelinase elevated the short-term facilitation of neurotransmitter release at the onset of each subsequent episode at 10 Hz (but not at 20 Hz) stimulation. Experiments with a combination of electrophysiological detection of evoked postsynaptic responses, monitoring of exocytotic release of the FM1-43 dye, and the use of FM1-43 fluorescence quencher that penetrates through the fusion pores have demonstrated that a significant portion of synaptic vesicles release the neurotransmitter through the transient fusion pore (kiss-and-run mechanism) during high-frequency (70 Hz) activity [4, 5]. Treatment with sphingomyelinase inhibited the neurotransmitter release pathway. As a consequence, the majority of neurosecretion events went through exocytosis, completely incorporating the vesicular membrane into the presynaptic membrane. Sphingomyelin in the membranes of presynaptic and synaptic vesicles is believed to have varying functions in regulating neurotransmitter release. To investigate this theory, we stimulated the exo-endocytosis processes of synaptic vesicles using sphingomyelinase, which provided access to the synaptic vesicle membranes. Under these circumstances, the enhancing impact of sphingomyelinase on neurotransmitter release and FM1-43 exocytosis rate were notably inhibited. Based on the obtained data, we hypothesize that sphingomyelinase disrupts the integrity of lipid rafts and selectively forms ceramide accumulations in synaptic membranes within the muscles at low concentrations. This ultimately leads to an increase in the mobilization of synaptic vesicles towards exocytosis sites during 10–70 Hz activity. Presumably, the synaptic vesicles from a distinct pool, which predominantly mediates the neurotransmission at a frequency of 10 Hz, are the most actively involved in exocytosis under these circumstances. Sphingomyelinase modifies membrane properties and can inhibit the release of neurotransmitters through the fusion pore during high-frequency activity, redirecting exocytosis towards the complete fusion pathway. Hydrolysis of sphingomyelin in both plasma and synaptic vesicle membranes reduces the stimulatory effects of sphingomyelinase, indicating a contrasting role of sphingomyelin in presynaptic and vesicular membranes for controlling neurotransmitter release [5].

Protein phosphorylation in isolated plasma membranes and postsynaptic junctional structures from brain synapses

In tauopathies, such as Alzheimer’s disease (AD), oligomeric tau (oTau) can induce tau hyperphosphorylation and further oligomerization, contributing to dendrite and dendritic spine degeneration. The p75 neurotrophin receptor (p75NTR), which regulates signaling that overlaps with tauopathy-related degenerative pathways, is a promising therapeutic target. LM11A-31, a small-molecule p75NTR modulator, downregulates p75NTR’s degenerative signaling and upregulates signaling that promotes dendritic and synaptic integrity. In a tauopathy mouse model, LM11A-31 reversed spine loss in hippocampal pyramidal neurons and improved performance on behavioral tests of hippocampal function. In a Phase 2a randomized, placebo-controlled AD clinical trial, LM11A-31 slowed the progression of synaptic degeneration biomarkers. In the present study, we tested the hypothesis that LM11A-31 enhances resilience of spines and dendrites to pathological forms of tau and/or inhibits the formation and accumulation of pathological tau species. We found that LM11A-31 prevented degeneration of spines and dendrites in cultured hippocampal neurons exposed to recombinant oTau or oTau-containing hippocampal extract fractions from PS19 tauopathy mice. Treating PS19 mice with LM11A-31 reduced the degeneration-promoting activity in oTau-containing hippocampal extract fractions. In cultured hippocampal neurons, LM11A-31 inhibited excess tau phosphorylation and aggregation induced by oTau and counteracted oTau-induced abnormalities in RhoA, PKC, LIMK1, and cofilin signaling, a signaling module known to regulate spine integrity. The ability of LM11A-31 to confer resilience to dendritic spines was partially dependent on LIMK1 activity. These findings demonstrate that small-molecule modulation of p75NTR by LM11A-31 both reduces the accumulation of pathological tau species and promotes resilience against oTau-induced dendritic spine degeneration, exemplifying a “dual-mechanism” therapeutic approach for tauopathies.

Amplifying mitochondrial function rescues adult neurogenesis in a mouse model of Alzheimer's disease

The role of the drebrin/EB3/Cdk5 pathway in dendritic spine plasticity, implications for Alzheimer's disease.

Dendritic spine morphology is associated with the current state of the synapse and neuron, and changes during synaptic plasticity in response to stimulus. At the same time, dendritic spine alterations are reported during various neurodegenerative and neurodevelopmental disorders and other brain states. Accurate and informative analysis of spine shape has an urgent need for studying the synaptic processes and molecular pathways in normal and pathological conditions, and for testing synapto-protective strategies during preclinical studies. Primary neuronal cultures enable high quality imaging of dendritic spines and offer a wide spectrum of accessible experimental manipulations. This article outlines the protocol for isolating, culturing, fluorescent labeling, and imaging of mouse primary hippocampal neurons by three-dimensional (3D) confocal microscopy in a normal state and in conditions of low amyloid toxicity-an in vitro model of Alzheimer's disease. An alternate protocol describes the neuronal morphology analysis using the EGFP expressing neurons in line-M transgenic mouse brain slices. Since the dendritic spines are relatively small structures lying close to the confocal microscope resolution limit, their proper segmentation on the images is challenging. This protocol highlights the image-preprocessing steps, including generation of theoretical point spread function and deconvolution, which enhances resolution and removes noise, thereby enhancing the 3D spine reconstruction results. SpineTool, an open source Python-based script, enables 3D segmentation of dendrites and spines and numerical metric calculation, including key measures, such as spine length, volume, and surface area, with a new feature, the chord length distribution histogram, improving clustering results. SpineTool supports both manual and machine learning spine classification (i.e., mushroom, thin, stubby, filopodia) and automated clustering using k-means and DBSCAN methods. This protocol provides detailed instructions for using SpineTool to analyze and classify dendritic spines in control and experimental groups, enhancing our understanding of spine morphology across different experimental conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Obtaining 3D confocal dendritic spine images of hippocampal neuronal culture in normal state and conditions of low amyloid toxicity Alternate Protocol: Obtaining confocal dendritic spine images of mice hippocampal neurons from fixed brain slices Support Protocol: Post-processing deconvolution of confocal images Basic Protocol 2: Segmentation of dendritic spines with SpineTool Basic Protocol 3: Spine dataset preparation using SpineTool Basic Protocol 4: Clustering of dendritic spines with SpineTool Basic Protocol 5: Machine classification of dendritic spines with SpineTool.

Application of in Vivo Fluorescence Imaging and Metal Ion Detection for Investigation of Bis(ethylmaltolato) Oxidovanadium (IV) on Alzheimer's Disease

Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia in the elderly. Accumulating evidence supports soluble amyloid-β (Aβ) oligomers as the leading candidate for the causative agent in AD and synapses as the primary site of Aβ oligomer action. However, the molecular and cellular mechanisms by which Aβ oligomers cause synaptic dysfunction and cognitive impairments remain poorly understood. Using primary cultures of rat hippocampal neurons as a model system, we show that the partitioning defective-1 (PAR-1)/microtubule affinity-regulating kinase (MARK) family kinases act as critical mediators of Aβ toxicity on synapses and dendritic spines. Overexpression of MARK4 led to tau hyperphosphorylation, reduced expression of synaptic markers, and loss of dendritic spines and synapses, phenotypes also observed after Aβ treatment. Importantly, expression of a non-phosphorylatable form of tau with the PAR-1/MARK site mutated blocked the synaptic toxicity induced by MARK4 overexpression or Aβ treatment. To probe the involvement of endogenous MARK kinases in mediating the synaptic toxicity of Aβ, we employed a peptide inhibitor capable of effectively and specifically inhibiting the activities of all PAR-1/MARK family members. This inhibitor abrogated the toxic effects of Aβ oligomers on dendritic spines and synapses as assayed at the morphological and electrophysiological levels. Our results reveal a critical role for PAR-1/MARK kinases in AD pathogenesis and suggest PAR-1/MARK inhibitors as potential therapeutics for AD and possibly other tauopathies where aberrant tau hyperphosphorylation is involved.

Researchers find that two microRNAs prime the brain to learn.

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis.SIGNIFICANCE STATEMENT Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

Alzheimer's disease (AD) is the most common cause of dementia among elderly people. Despite enormous efforts, the pathogenesis of AD still remains unclear and no drug has yet been proved to be disease-modifying. As the basis of learning and memory, the plasticity of synapse and dendritic spine has been impaired during AD progression. Previous studies have showed a protective effect of L-3-n-butylphthalide (L-NBP) on cognitive deficits in AD, we wonder whether this protective effect is associated with positive alterations on synapse and dendritic spines. In this study, we first of all confirmed the anti-dementia effect of L-NBP in 13-month-old APP/PS1 mice, and then investigated the alterations in synaptic and dendritic spine plasticity due to L-NBP treatment both in vivo and in vitro. We also conducted preliminary studies and found the possible mechanisms related to the inhibition of over-activated complement cascade and the remodeling of actin cytoskeleton. Besides, we also found extra benefits of L-NBP on presynaptic dystrophic neurites and attempted to give explanations from the view of autophagy regulation. Taken together, our study added some new evidence to the application of L-NBP in AD treatment and provided deeper insight into the relevant mechanisms for future study.