A brain atlas of synapse protein lifetime across the mouse lifespan.

Protein turnover is required for synapse maintenance and remodelling and may impact memory duration. We quantified the lifetime of postsynaptic protein PSD95 in individual excitatory synapses across the mouse brain and lifespan, generating the Protein Lifetime Synaptome Atlas. Excitatory synapses have a wide range of protein lifetimes that may extend from a few hours to several months, with distinct spatial distributions in dendrites, neuron types and brain regions. Short protein lifetime (SPL) synapses are enriched in developing animals and in regions controlling innate behaviors, whereas long protein lifetime (LPL) synapses accumulate during development, are enriched in the cortex and CA1 where memories are stored, and are preferentially preserved in old age. The protein lifetime synaptome architecture is disrupted in an autism model, with synapse protein lifetime increased throughout the brain. These findings add a further layer to synapse diversity in the brain and enrich prevailing concepts in behavior, development, ageing and brain repair.

Contribution of Membrane Lipids to Postsynaptic Protein Organization

The precise subsynaptic organization of proteins at the postsynaptic membrane controls synaptic transmission. In particular, postsynaptic receptor complexes are concentrated in distinct membrane nanodomains to optimize synaptic signaling. However, despite the clear functional relevance of subsynaptic receptor organization to synaptic transmission and plasticity, the mechanisms that underlie the nanoscale organization of the postsynaptic membrane remain elusive. Over the last decades, the field has predominantly focused on the role of protein-protein interactions in receptor trafficking and positioning in the synaptic membrane. In contrast, the contribution of lipids, the principal constituents of the membrane, to receptor positioning at the synapse remains poorly understood. Nevertheless, there is compelling evidence that the synaptic membrane is enriched in specific lipid species and that deregulation of lipid homeostasis in neurons severely affects synaptic functioning. In this review we focus on how lipids are organized at the synaptic membrane, with special emphasis on how current models of membrane organization could contribute to protein distribution at the synapse and synaptic transmission. Finally, we will present an outlook on how novel technical developments could be applied to study the dynamic interplay between lipids and proteins at the postsynaptic membrane.

Differential Expression Patterns of TDP-43 in Single Moderate versus Repetitive Mild Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) is a disabling disorder and a major cause of death and disability in the world. Both single and repetitive traumas affect the brain acutely but can also lead to chronic neurodegenerative changes. Clinical studies have shown some dissimilarities in transactive response DNA binding protein 43 (TDP-43) expression patterns following single versus repetitive TBI. We explored the acute cortical post-traumatic changes of TDP-43 using the lateral fluid percussion injury (LFPI) model of single moderate TBI in adult male mice and investigated the association of TDP-43 with post-traumatic neuroinflammation and synaptic plasticity. In the ipsilateral cortices of animals following LFPI, we found changes in the cytoplasmic and nuclear levels of TDP-43 and the decreased expression of postsynaptic protein 95 within the first 3 d post-injury. Subacute pathological changes of TDP-43 in the hippocampi of animals following LFPI and in mice exposed to repetitive mild TBI (rmTBI) were studied. Changes in the hippocampal TDP-43 expression patterns at 14 d following different brain trauma procedures showed pathological alterations only after single moderate, but not following rmTBI. Hippocampal LFPI-induced TDP-43 pathology was not accompanied by the microglial reaction, contrary to the findings after rmTBI, suggesting that different types of brain trauma may cause diverse pathophysiological changes in the brain, specifically related to the TDP-43 protein as well as to the microglial reaction. Taken together, our findings may contribute to a better understanding of the pathophysiological events following brain trauma.

Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line

A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50–60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.

A-8 Sex Modifies the Association between CSF Neurogranin and Cognitive Decline in Older Adults

Objective Neurogranin is a postsynaptic protein associated with declining memory and executive functioning in Alzheimer’s disease (ad). While previous research suggests neurogranin concentrations in ad are higher in women, it is unclear whether sex differences exist in earlier disease stages or predict different cognitive outcomes. This study investigates cerebrospinal fluid (CSF) neurogranin in relation to longitudinal cognitive decline in older adults ranging from normal cognition to mild cognitive impairment, assessing for interactions by sex. Method Vanderbilt Memory & Aging Project participants completed baseline fasting lumbar puncture (n = 155, 73 ± 8 years) for neurogranin quantification and serial neuropsychological assessments at 18-month intervals. Linear mixed effect regression adjusting for age, sex (for main effect models), race/ethnicity, education, cognitive diagnosis, depressed mood, and APOE-ε4 carrier status. Results CSF neurogranin predicted worse cognitive decline across multiple domains (p-values <0.05). Sex interactions existed for Boston Naming Test (β = −0.007, p = 0.001), WAIS-IV Coding (β = −0.01, p = 0.02), Hooper Visual Orientation Test (β = −0.005, p = 0.02), and Category (animals) Fluency (β = −0.005, p = 0.048) wherein CSF neurogranin predicted worse decline among women (p-values≤0.03) but not men (p-values≥0.36). Conclusion Results suggest that among nondemented older adults, CSF neurogranin predicts worse longitudinal cognitive decline in women but not in men. Further research is needed to elucidate underlying mechanisms that may account for these differences, such as possible sex hormone factors. These findings highlight the importance of pursuing individualized prevention and treatment approaches to combat accelerated cognitive aging that take into account the possibility of multiple, divergent disease pathways preceding ad and dementia among various demographic groups.

Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.

The Role of Stress-Induced Changes of Homer1 Expression in Stress Susceptibility

Stress negatively affects processes of synaptic plasticity and is a major risk factor of various psychopathologies such as depression and anxiety. HOMER1 is an important component of the postsynaptic density: constitutively expressed long isoforms HOMER1b and HOMER1c bind to group I metabotropic glutamate receptors MGLUR1 (GRM1) and MGLUR5 and to other effector proteins, thereby forming a postsynaptic protein scaffold. Activation of the GLUR1–HOMER1b,c and/or GLUR5–HOMER1b,c complex regulates activity of the NMDA and AMPA receptors and Ca2+ homeostasis, thus modulating various types of synaptic plasticity. Dominant negative transcript Homer1a is formed as a result of activity-induced alternative termination of transcription. Expression of this truncated isoform in response to neuronal activation impairs interactions of HOMER1b,c with adaptor proteins, triggers ligand-independent signal transduction through MGLUR1 and/or MGLUR5, leads to suppression of the AMPA- and NMDA-mediated signal transmission, and thereby launches remodeling of the postsynaptic protein scaffold and inhibits long-term potentiation. The studies on animal models confirm that the HOMER1a-dependent remodeling most likely plays an important part in the stress susceptibility, whereas HOMER1a itself can be regarded as a neuroprotector. In this review article, we consider the effects of different stressors in various animal models on HOMER1 expression as well as impact of different HOMER1 variants on human behavior as well as structural and functional characteristics of the brain.

An optogenetic method for investigating presynaptic molecular regulation

While efficient methods are well established for studying postsynaptic protein regulation of glutamatergic synapses in the mammalian central nervous system, similarly efficient methods are lacking for studying proteins regulating presynaptic function. In the present study, we introduce an optical/electrophysiological method for investigating presynaptic molecular regulation. Here, using an optogenetic approach, we selectively stimulate genetically modified presynaptic CA3 pyramidal neurons in the hippocampus and measure optically-induced excitatory postsynaptic currents produced in unmodified postsynaptic CA1 pyramidal neurons. While such use of optogenetics is not novel, previous implementation methods do not allow basic quantification of the changes in synaptic strength produced by genetic manipulations. We find that incorporating simultaneous recordings of fiber volley amplitude provides a control for optical stimulation intensity and, as a result, creates a metric of synaptic efficacy that can be compared across experimental conditions. In the present study, we utilize our new method to demonstrate that inhibition of synaptotagmin 1 expression in CA3 pyramidal neurons leads to a significant reduction in Schaffer collateral synapse function, an effect that is masked with conventional electrical stimulation. Our hope is that this method will expedite our understanding of molecular regulatory pathways that govern presynaptic function.

Low-level Chlorpyrifos Exposure Affects Synaptic Plasticity in Hippocampal Neurons

Chlorpyrifos (CPF) is a widely used organophosphorus pesticide and exhibits environmental persistence and bioaccumulation. Increasing evidence has shown that exposure to CPF in early life might induce neurodevelopmental disorders in adulthood. Synaptic plasticity plays a crucial role in neurodevelopment. This study aimed to investigate the effect of CPF on synaptic plasticity in hippocampal neurons and establish the cellular mechanism of these effects. We analysed the impact of CPF on the expression level of a presynaptic protein, a postsynaptic protein, and ionotropic glutamate receptors, as well as the effects on the Wnt7a/β-catenin pathway in primary hippocampal neurons. We found that CPF decreased the expression of synaptophysin, PSD-95, NMDAR1, GluR1, and Wnt7a, as well as active β-catenin, in primary hippocampal neurons. Our study suggests that low-level CPF exposure affects synaptic plasticity in hippocampal neurons.

Full-length and C-terminal neurogranin in Alzheimer’s disease cerebrospinal fluid analyzed by novel ultrasensitive immunoassays

Background Neurogranin (Ng) is a neuron-specific and postsynaptic protein that is abundantly expressed in the brain, particularly in the dendritic spine of the hippocampus and cerebral cortex. The enzymatic cleavage of Ng produces fragments that are released into cerebrospinal (CSF), which have been shown to be elevated in Alzheimer’s disease (AD) patients and predict cognitive decline. Thus, quantification of distinctive cleavage products of Ng could elucidate different features of the disease. Methods In this study, we developed novel ultrasensitive single molecule array (Simoa) assays for measurement of full-length neurogranin (FL-Ng) and C-terminal neurogranin (CT-Ng) fragments in CSF. The Ng Simoa assays were evaluated in CSF samples from AD patients (N = 23), mild cognitive impairment due to AD (MCI-AD) (N = 18), and from neurological controls (N = 26). Results The intra-assay repeatability and inter-assay precision of the novel methods had coefficients of variation below 7% and 14%, respectively. CSF FL-Ng and CSF CT-Ng median concentrations were increased in AD patients (6.02 ng/L, P < 0.00001 and 452 ng/L, P = 0.00001, respectively) and in patients with MCI-AD (5.69 ng/L, P < 0.00001 and 566 ng/L, P < 0.00001) compared to neurological controls (0.644 ng/L and 145 ng/L). The median CSF ratio of CT-Ng/FL-Ng were decreased in AD patients (ratio = 101, P = 0.008) and in patients with MCI-AD (ratio = 115, P = 0.016) compared to neurological controls (ratio = 180). CSF of FL-Ng, CT-Ng, and ratio of CT-Ng/FL-Ng could each significantly differentiate AD patients from controls (FL-Ng, AUC = 0.907; CT-Ng, AUC = 0.913; CT-Ng/FL-Ng, AUC = 0.775) and patients with MCI-AD from controls (FL-Ng, AUC = 0.937; CT-Ng, AUC = 0.963; CT-Ng/FL-Ng, AUC = 0.785). Conclusions Assessments of the FL-Ng and CT-Ng levels in CSF with the novel sensitive immunoassays provide a high separation of AD from controls, even in early phase of the disease. The novel Ng assays are robust and highly sensitive and may be valuable tools to study synaptic alteration in AD, as well as to monitor the effect on synaptic integrity of novel drug candidates in clinical trials.