synaptic markers
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2022 ◽  
Author(s):  
Francesco Limone ◽  
Jana M. Mitchell ◽  
Irune Guerra San Juan ◽  
Janell L.M. Smith ◽  
Kavya Raghunathan ◽  
...  

Human pluripotent stem cells (hPSCs) are a powerful tool for disease modelling and drug discovery, especially when access to primary tissue is limited, such as in the brain. Current neuronal differentiation approaches use either small molecules for directed differentiation or transcription-factor-mediated programming. In this study we coupled the overexpression of the neuralising transcription factor Neurogenin2 (Ngn2) with small molecule patterning to differentiate hPSCs into lower induced Motor Neurons (liMoNes). We showed that this approach induced activation of the motor neuron (MN) specific transcription factor Hb9/MNX1, using an Hb9::GFP-reporter line, with up to 95% of cells becoming Hb9::GFP+. These cells acquired and maintained expression of canonical early and mature MN markers. Molecular and functional profiling revealed that liMoNes resembled bona fide hPSC-derived MN differentiated by conventional small molecule patterning. liMoNes exhibited spontaneous electrical activity, expressed synaptic markers and formed contacts with muscle cells in vitro. Pooled, multiplex single-cell RNA sequencing on 50 cell lines revealed multiple anatomically distinct MN subtypes of cervical and brachial, limb-innervating MNs in reproducible quantities. We conclude that combining small molecule patterning with Ngn2 can facilitate the high-yield, robust and reproducible production of multiple disease-relevant MN subtypes, which is fundamental in the path to propel forward our knowledge of motoneuron biology and its disruption in disease.


2022 ◽  
Vol 15 ◽  
Author(s):  
Joe K. Chouhan ◽  
Ursula Püntener ◽  
Steven G. Booth ◽  
Jessica L. Teeling

Bacterial infections are a common cause of morbidity and mortality in the elderly, and particularly in individuals with a neurodegenerative disease. Experimental models of neurodegeneration have shown that LPS-induced systemic inflammation increases neuronal damage, a process thought to be mediated by activation of “primed” microglia. The effects of a real systemic bacterial infection on the innate immune cells in the brain and neuronal networks are less well described, and therefore, in this study we use the ME7 prion model to investigate the alterations in microglia activation and phenotype and synaptic markers in response to a low grade, live bacterial infection. Mice with or without a pre-existing ME7 prion-induced neurodegenerative disease were given a single systemic injection of live Salmonella typhimurium at early or mid-stage of disease progression. Immune activation markers CD11b and MHCII and pro-inflammatory cytokines were analyzed 4 weeks post-infection. Systemic infection with S. typhimurium resulted in an exaggerated inflammatory response when compared to ME7 prion mice treated with saline. These changes to inflammatory markers were most pronounced at mid-stage disease. Analysis of synaptic markers in ME7 prion mice revealed a significant reduction of genes that are associated with early response in synaptic plasticity, extracellular matrix structure and post-synaptic density, but no further reduction following systemic infection. In contrast, analysis of activity-related neuronal receptors involved in development of learning and memory, such as Grm1 and Grin2a, showed a significant decrease in response to systemic bacterial challenge. These changes were observed early in the disease progression and associated with reduced burrowing activity. The exaggerated innate immune activation and altered expression of genes linked to synaptic plasticity may contribute to the onset and/or progression of neurodegeneration.


Biomolecules ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 34
Author(s):  
Leticia Pérez-Sisqués ◽  
Júlia Solana-Balaguer ◽  
Genís Campoy-Campos ◽  
Núria Martín-Flores ◽  
Anna Sancho-Balsells ◽  
...  

RTP801/REDD1 is a stress-regulated protein whose levels are increased in several neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s diseases (HD). RTP801 downregulation ameliorates behavioral abnormalities in several mouse models of these disorders. In HD, RTP801 mediates mutant huntingtin (mhtt) toxicity in in vitro models and its levels are increased in human iPSCs, human postmortem putamen samples, and in striatal synaptosomes from mouse models of the disease. Here, we investigated the role of RTP801 in the hippocampal pathophysiology of HD. We found that RTP801 levels are increased in the hippocampus of HD patients in correlation with gliosis markers. Although RTP801 expression is not altered in the hippocampus of the R6/1 mouse model of HD, neuronal RTP801 silencing in the dorsal hippocampus with shRNA containing AAV particles ameliorates cognitive alterations. This recovery is associated with a partial rescue of synaptic markers and with a reduction in inflammatory events, especially microgliosis. Altogether, our results indicate that RTP801 could be a marker of hippocampal neuroinflammation in HD patients and a promising therapeutic target of the disease.


2021 ◽  
Author(s):  
Celia Luchena ◽  
Jone Zuazo-Ibarra ◽  
Jorge Valero ◽  
Carlos Matute ◽  
Elena Alberdi ◽  
...  

Glial cells are essential to understand Alzheimer disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated a triple co-culture with neurons, microglia and astrocytes. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aβ (oAβ) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory markers Arginase I and TGF-β1, and reduced pro-inflammatory iNOS and IL-1β, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAβ in the triple co-culture reduced synaptic markers and increased microglial activation, which are hallmarks of AD. Consequently, we developed a reliable model, where cells better resemble physiological conditions: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology than in individual primary cultures. Moreover, we were able to recapitulate Aβ-induced synaptic loss and inflammation. This model emerges as a powerful tool to study neurodegeneration and inflammation in the context of AD and other neurodegenerative diseases.


2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Beatriz G. Perez-Nievas ◽  
Louisa Johnson ◽  
Paula Beltran-Lobo ◽  
Martina M. Hughes ◽  
Luciana Gammallieri ◽  
...  

Abstract Background Pathological interactions between β-amyloid (Aβ) and tau drive synapse loss and cognitive decline in Alzheimer’s disease (AD). Reactive astrocytes, displaying altered functions, are also a prominent feature of AD brain. This large and heterogeneous population of cells are increasingly recognised as contributing to early phases of disease. However, the contribution of astrocytes to Aβ-induced synaptotoxicity in AD is not well understood. Methods We stimulated mouse and human astrocytes with conditioned medium containing concentrations and species of human Aβ that mimic those in human AD brain. Medium from stimulated astrocytes was collected and immunodepleted of Aβ before being added to naïve rodent or human neuron cultures. A cytokine, identified in unbiased screens of stimulated astrocyte media and in postmortem human AD brain lysates was also applied to neurons, including those pre-treated with a chemokine receptor antagonist. Tau mislocalisation, synaptic markers and dendritic spine numbers were measured in cultured neurons and organotypic brain slice cultures. Results We found that conditioned medium from stimulated astrocytes induces exaggerated synaptotoxicity that is recapitulated following spiking of neuron culture medium with recombinant C–X–C motif chemokine ligand-1 (CXCL1), a chemokine upregulated in AD brain. Antagonism of neuronal C–X–C motif chemokine receptor 2 (CXCR2) prevented synaptotoxicity in response to CXCL1 and Aβ-stimulated astrocyte secretions. Conclusions Our data indicate that astrocytes exacerbate the synaptotoxic effects of Aβ via interactions of astrocytic CXCL1 and neuronal CXCR2 receptors, highlighting this chemokine–receptor pair as a novel target for therapeutic intervention in AD.


Author(s):  
Leticia Pérez-Sisqués ◽  
Júlia Solana-Balaguer ◽  
Genís Campoy-Campos ◽  
Núria Martín-Flores ◽  
Anna Sancho-Balsells ◽  
...  

RTP801/REDD1 is a stress-regulated protein whose levels are increased in several neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s diseases (HD). RTP801 downregulation ameliorates behavioral abnormalities in several mouse models of these disorders. In HD, RTP801 mediates mutant huntingtin (mhtt) toxicity in in vitro models and its levels are increased in human iPSCs, human postmortem putamen samples and in striatal synaptosomes from mouse models of the disease. Here, we investigated the role of RTP801 in the hippocampal pathophysiology of HD. We found that RTP801 levels are increased in the hippocampus of HD patients in correlation with gliosis markers. Although RTP801 expression is not altered in the hippocampus of the R6/1 mouse model of HD, neuronal RTP801 silencing in the dorsal hippocampus with shRNA-containing AAV particles ameliorates cognitive alterations. This recovery is associated with a partial rescue of synaptic markers and with a reduction of inflammatory events, especially microgliosis. Altogether, our results indicate that RTP801 could be a marker of hippocampal neuroinflammation in HD patients and a promising therapeutic target of the disease.


Author(s):  
Kirupa Suthakar ◽  
M. Charles Liberman

Cochlear synaptopathy is the noise-induced or age-related loss of ribbon synapses between inner hair cells (IHCs) and auditory nerve fibers (ANFs), first reported in CBA/CaJ mice. Recordings from single ANFs in anaesthesized, noise-exposed guinea pigs suggested that neurons with low spontaneous rates (SRs) and high thresholds are more vulnerable than low-threshold, high-SR fibers. However, there is extensive post-exposure regeneration of ANFs in guinea pigs, but not in mice. Here, we exposed CBA/CaJ mice to octave-band noise and recorded sound-evoked and spontaneous activity from single ANFs at least 2 weeks later. Confocal analysis of cochleae immunostained for pre- and post-synaptic markers confirmed the expected loss of 40 - 50% of ANF synapses in the basal half of the cochlea, however, our data were not consistent with a selective loss of low-SR fibers. Rather they suggested a loss of both SR groups in synaptopathic regions. Single-fiber thresholds and frequency tuning recovered to pre-exposure levels however, response to tone bursts showed increased peak and steady-state firing rates as well as decreased jitter in first-spike latencies. This apparent gain-of-function increased the robustness of tone-burst responses in the presence of continuous masking noise. This study suggests that the nature of noise-induced synaptic damage varies between different species and that, in mouse, the noise-induced hyperexcitability seen in central auditory circuits is also observed at the level of the auditory nerve.


Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2528
Author(s):  
Ilenia Martinelli ◽  
Daniele Tomassoni ◽  
Proshanta Roy ◽  
Francesco Amenta ◽  
Seyed Khosrow Tayebati

The association between obesity and loss of cognitive performance has been recognized. Although there are data regarding the metabolic alterations in obese conditions and the development of neuroinflammation, no clear evidence concerning obesity-related cholinergic and synaptic impairments in the frontal cortex and hippocampus has been reported yet. Here, we investigate different cholinergic and synaptic markers in 12-, 16-, and 20-week-old obese Zucker rats (OZRs) compared with lean littermate rats (LZRs), using immunochemical and immunohistochemical analysis. Consequently, OZRs showed body weight gain, hypertension, and dysmetabolism. In 20-week-old OZRs, the reduction of vesicular acetylcholine transporter (VAChT) and alpha7 nicotinic acetylcholine receptors (α7nAChR) occurred both in the frontal cortex and in the hippocampus, suggesting a cognitive dysfunction due to obesity and aging. Among the muscarinic receptors analyzed, the level of expression of type 1 (mAChR1) was lower in the hippocampus of the older OZRs. Finally, we showed synaptic dysfunctions in OZRs, with a reduction of synaptophysin (SYP) and synaptic vesicle glycoprotein 2B (SV2B) in 20-week-old OZRs, both in the frontal cortex and in the hippocampus. Taken together, our data suggest specific alterations of cholinergic and synaptic markers that can be targeted to prevent cognitive deficits related to obesity and aging.


2021 ◽  
Author(s):  
Triana Espinosa-Jimenez ◽  
Oriol Busquets ◽  
Amanda Cano ◽  
Ester Verdaguer ◽  
Jordi Olloquequi ◽  
...  

Abstract BackgroundPPARβ/δ, the most PPAR abundant isotype in the central nervous system is involved in the modulation of microglial homeostasis and metabolism. Several studies have demonstrated that people suffering from type 2 diabetes mellitus develop cognitive decline turning insulin resistance one of the best predictors of this disturbance. Although numerous investigations have studied the role of PPARb/d in metabolism, its role in neuronal and cognitive function has been underexplored. Therefore, the aim of the study is to determine the role of PPARb/d in the neuropathological pathways involved in the development of cognitive decline and as to whether a risk factor involved in cognitive loss such as obesity modulates neuropathological markers.6-month-old male PPARβ/δ-null (PPARβ/δ-/-) and wildtype (WT) littermates with the same genetic background (C57BL/6X129/SV) and exceptionally, C57BL/6 were used. After the weaning, animals were fed either with conventional chow (CT) or with a palmitic acid-enriched diet containing 45% of fat mainly from hydrogenated coconut oil (HFD). Thus, four groups were defined: WT CT, WT HFD, PPARβ/δ-/- CT and PPARβ/δ-/- HFD and several pathological mechanisms involved in cognitive decline were analyzed.ResultsOur results confirmed that C57BL/6X129/SV showed significantly increased levels of anxiety compared to C57BL/6. Therefore, to evaluate cognitive decline, behavioral tests were dismissed, and dendritic spine quantification and other biochemical biomarkers were performed.PPARβ/δ-/- mice exhibited a decrease in dendritic spine density and synaptic markers, suggesting an alteration in cognitive function and synaptic plasticity. Likewise, our study demonstrated that the lack of PPARβ/δ receptor enhances gliosis in the hippocampus, contributing to astrocyte and microglial activation and also induced an increase in neuroinflammatory biomarkers. Additionally, alterations in the hippocampal insulin receptor pathway were found. Interestingly, while some of the disturbances caused by the lack of PPARβ/δ were not affected by feeding the HFD, others were exacerbated or required the combination of both factors.ConclusionsTaken together, these findings suggest that the loss of PPARβ/δ-/- affects neuronal and synaptic structure, contributing to cognitive dysfunction and, they also present this receptor as a possible new target for the treatment of cognitive decline.


2021 ◽  
Vol 22 (17) ◽  
pp. 9583
Author(s):  
Sareer Ahmad ◽  
Myeung Hoon Jo ◽  
Muhammad Ikram ◽  
Amjad Khan ◽  
Myeong Ok Kim

The current study was undertaken to unveil the protective effects of Luteolin, a natural flavonoid, against amyloid-beta (Aβ1–42)-induced neuroinflammation, amyloidogenesis, and synaptic dysfunction in mice. For the development of an AD mouse model, amyloid-beta (Aβ1–42, 5 µL/5 min/mouse) oligomers were injected intracerebroventricularly (i.c.v.) into mice’s brain by using a stereotaxic frame. After that, the mice were treated with Luteolin for two weeks at a dose of 80 mg/kg/day. To monitor the biochemical changes, we conducted western blotting and immunofluorescence analysis. According to our findings, the infusion of amyloid-beta activated c-Jun N-terminal kinases (p-JNK), p38 mitogen-activated protein kinases, glial fibrillary acidic protein (GFAP), and ionized calcium adaptor molecule 1 (Iba-1) in the cortex and hippocampus of the experimental mice; these changes were significantly inhibited in Aβ1–42 + Luteolin-treated mice. Likewise, we also checked the expression of inflammatory markers, such as p-nuclear factor-kB p65 (p-NF-kB p65 (Ser536), tissue necrosis factor (TNF-α), and Interleukin1-β (IL-1β), in Aβ1–42-injected mice brain, which was attenuated in Aβ1–42 + Luteolin-treated mice brains. Further, we investigated the expression of pro- and anti-apoptotic cell death markers such as Bax, Bcl-2, Caspase-3, and Cox-2, which was significantly reduced in Aβ1–42 + Lut-treated mice brains compared to the brains of the Aβ-injected group. The results also indicated that with the administration of Aβ1–42, the expression levels of β-site amyloid precursor protein cleaving enzyme (BACE-1) and amyloid-beta (Aβ1–42) were significantly enhanced, while they were reduced in Aβ1–42 + Luteolin-treated mice. We also checked the expression of synaptic markers such as PSD-95 and SNAP-25, which was significantly enhanced in Aβ1–42 + Lut-treated mice. To unveil the underlying factors responsible for the protective effects of Luteolin against AD, we used a specific JNK inhibitor, which suggested that Luteolin reduced Aβ-associated neuroinflammation and neurodegeneration via inhibition of JNK. Collectively, our results indicate that Luteolin could serve as a novel therapeutic agent against AD-like pathological changes in mice.


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