Synaptic actions of amyotrophic-lateral-sclerosis-associated G85R-SOD1 in the squid giant synapse

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Song, Y. Synaptic actions of amyotrophic-lateral-sclerosis-associated G85R-SOD1 in the squid giant synapse. Eneuro, (2020): ENEURO.0369-19.2020, doi: 10.1523/ENEURO.0369-19.2020.Altered synaptic function is thought to play a role in many neurodegenerative diseases, but little is known about the underlying mechanisms for synaptic dysfunction. The squid giant synapse (SGS) is a classical model for studying synaptic electrophysiology and ultrastructure, as well as molecular mechanisms of neurotransmission. Here, we conduct a multidisciplinary study of synaptic actions of misfolded human G85R-SOD1 causing familial Amyotrophic Lateral Sclerosis (fALS). G85R-SOD1, but not WT-SOD1, inhibited synaptic transmission, altered presynaptic ultrastructure, and reduced both the size of the Readily Releasable Pool (RRP) of synaptic vesicles and mobility from the Reserved Pool (RP) to the RRP. Unexpectedly, intermittent high frequency stimulation (iHFS) blocked inhibitory effects of G85R-SOD1 on synaptic transmission, suggesting aberrant Ca2+ signaling may underlie G85R-SOD1 toxicity. Ratiometric Ca2+ imaging showed significantly increased presynaptic Ca2+ induced by G85R-SOD1 that preceded synaptic dysfunction. Chelating Ca2+ using EGTA prevented synaptic inhibition by G85R-SOD1, confirming the role of aberrant Ca2+ in mediating G85R-SOD1 toxicity. These results extended earlier findings in mammalian motor neurons and advanced our understanding by providing possible molecular mechanisms and therapeutic targets for synaptic dysfunctions in ALS as well as a unique model for further studies.Grass Foundation, HHMI, MGH Jack Satter Foundation, Harvard University ALS and Alzheimer's Endowed Research Fund, Harvard Brain Science Initiative

Image informatics strategies for deciphering neuronal network connectivity

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies

Fluid Biomarkers for Synaptic Dysfunction and Loss

Synapses are the site for brain communication where information is transmitted between neurons and stored for memory formation. Synaptic degeneration is a global and early pathogenic event in neurodegenerative disorders with reduced levels of pre- and postsynaptic proteins being recognized as a core feature of Alzheimer’s disease (AD) pathophysiology. Together with AD, other neurodegenerative and neurodevelopmental disorders show altered synaptic homeostasis as an important pathogenic event, and due to that, they are commonly referred to as synaptopathies. The exact mechanisms of synapse dysfunction in the different diseases are not well understood and their study would help understanding the pathogenic role of synaptic degeneration, as well as differences and commonalities among them and highlight candidate synaptic biomarkers for specific disorders. The assessment of synaptic proteins in cerebrospinal fluid (CSF), which can reflect synaptic dysfunction in patients with cognitive disorders, is a keen area of interest. Substantial research efforts are now directed toward the investigation of CSF synaptic pathology to improve the diagnosis of neurodegenerative disorders at an early stage as well as to monitor clinical progression. In this review, we will first summarize the pathological events that lead to synapse loss and then discuss the available data on established (eg, neurogranin, SNAP-25, synaptotagmin-1, GAP-43, and α-syn) and emerging (eg, synaptic vesicle glycoprotein 2A and neuronal pentraxins) CSF biomarkers for synapse dysfunction, while highlighting possible utilities, disease specificity, and technical challenges for their detection

Synaptic loss in the primary tauopathies of Progressive Supranuclear Palsy and Corticobasal Degeneration

In this thesis I address the debilitating symptom of cognitive dysfunction in the primary tauopathies of Progressive Supranuclear Palsy (PSP) and Corticobasal Degeneration (CBD). Both PSP and CBD are associated with an accumulation of 4-repeat tau in cortical and subcortical areas. As well as movement disorders, they impair cognitive function, even where there is minimal atrophy. Neurophysiological studies have also identified electrophysiological changes associated with cognitive dysfunction, in areas without atrophy. I propose that synaptic loss prior to cell loss contributes to these effects of disease. Chapter two summarises my cohort and principal methods. I quantify synaptic density in vivo with dynamic [11C]UCB-J PET, and molecular pathology with [18F]AV1451 PET. Brain structural changes are quantified by MRI. Disease severity and cognition are assessed with the PSP rating scale, and neuropsychological tests. Patients with CBD are negative on amyloid-imaging ([11C]PiB PET) to exclude those with Alzheimer’s pathology. In chapter three, [11C]UCB-J PET reveals widespread loss of synapses in PSP and CBD including areas with minimal atrophy. The loss of synapses correlated with cognition and disease severity. In chapter four, I test whether presynaptic changes (from [11C]UCB-J PET) are correlated with postsynaptic abnormalities (i.e. changes to postsynaptic dendritic microstructural integrity quantified by MRI using the Neurite Orientation and Dispersion Index, NODDI). In accordance with in vitro and animal models, I confirm that loss of dendritic complexity is tightly coupled with presynaptic density, over and above the effects of atrophy. In chapter five, I test the relationship between the molecular pathology in primary tauopathies (tau burden) and synaptic loss, using [18F]AV-1451 and [11C]UCB-J PET. The use of the “tau” ligand [18F]AV-1451 has become controversial in PSP. With due consideration to the caveats, I report that brain regions with a higher synaptic density have higher [18F]AV-1451 binding, consistent with the hypothesis of connectivity-based progression of tauopathy. I further show that accrual of pathology in any given area is associated with loss of synapses, consistent with synaptic injury from tauopathy. I conclude my thesis in chapter 6, by discussing and highlighting the importance of synaptic density in primary tauopathies. The findings are relevant to other neurodegenerative disorders, and support early interventional studies targeting synaptic maintenance and restoration.Association of British Neurologists - Patrick Berthoud Charitable Trus

Formation and Characterization of in vitro Bioengineered Neuromuscular Junction Models

In vitro models of the neuromuscular junction (NMJ) are emerging as a valuable tool to study synaptogenesis, synaptic maintenance, and pathogenesis of neurodegenerative diseases. Many models have previously been developed using a variety of cell sources for muscle and motoneurons, but the models can be further improved by integrating beneficial features to better mimic the native milieu of NMJ development. We created a functional in vitro model of NMJ by bioreactor cultivation of C2C12 myoblasts, transdifferentiated myocytes and stem cell-derived motoneurons with electrical stimulation. Proper coculture medium and electrical stimulation led to improved functional coupling between the emerging motoneurons and myocytes, as evidenced by mature cellular structures, increased expression of neuronal and muscular genes, clusterization of acetylcholine receptors (AChRs) in the vicinity of motoneurons, and the response of the coculture to glutamate stimulation. To validate the models and demonstrate utility for pharmacological testing, we analyzed the potency of the drugs that affect key pathways during NMJ signal transduction, including acetylcholine (ACh) synthesis, ACh vesicular storage, ACh synaptic release, AChR activation, and ACh inactivation in the synaptic cleft. The models properly responded to the drugs in a concentration-dependent manner. The proposed in vitro NMJ model could thus be used in pharmacological screening and controlled studies of neuromuscular diseases

Dissection of synaptic pathways through the CSF biomarkers for predicting Alzheimer's disease

OBJECTIVE: To assess the ability of a combination of synaptic CSF biomarkers to separate AD and non-AD disorders and to help in the differential diagnosis between neurocognitive diseases. METHODS: Retrospective cross-sectional monocentric study. All participants explored with CSF assessments for neurocognitive decline were invited to participate. After complete clinical and imaging evaluations, 243 patients were included. CSF synaptic (GAP-43, neurogranin, SNAP-25 total, SNAP-25 aa40, synaptotagmin-1) and AD biomarkers were blindly quantified using ELISA or mass spectrometry. Statistical analysis compared CSF levels between various groups AD dementias n=81, MCI-AD n=30, other MCI n=49, other dementias (OD) n=49, neurological controls n=35) as well as their discriminatory powers. RESULTS: All synaptic biomarkers were significantly increased in MCI-AD and AD -dementias patients compared to other groups. All synaptic biomarkers could efficiently discriminate AD dementias from OD (AUC ≥0.80). All but synaptotagmin were also able to discriminate MCI-AD from controls (AUC ≥0.85) and AD dementias from controls (AUC ≥0.80). Overall, CSF SNAP 25aa40 had the highest discriminative power (AUC=0.93) between AD dementias and controls or OD, and AUC=0.90 between MCI-AD and controls. Higher levels were associated with two alleles of apolipoprotein E (APOE) ε4. CONCLUSION: All synaptic biomarkers tested had a good discriminatory power to distinguish patients with AD abnormal CSF from non-AD disorders. SNAP25aa40 demonstrated the highest power to discriminate AD CSF positive patients from non-AD patients and neurological controls in this cohort. CLASSIFICATION OF EVIDENCE: This retrospective study provides Class II evidence that CSF synaptic biomarkers discriminate patients with AD from non-AD patients

Quantitative Imaging of Net Axonal Transport in vivo: A Biomarker for Motor Neuron Health and Disease

Amyotrophic lateral sclerosis (ALS) is a lethal, progressive neurodegenerative disorder that selectively affects both upper and lower motor neurons, leading to muscle weakness, paralysis and death. Despite recent advances in the identification of genes associated with ALS, the quest for a sensitive biomarker for rapid and accurate diagnosis, prognosis, and treatment response monitoring has not been fulfilled. In this thesis, I report a method of quantifying the integrity of motor neurons in vivo using imaging to record uptake and retrograde transport of intramuscularly injected tetanus toxin fragment C (TTC) into spinal motor neurons. This method tracks and profiles progression of disease (transgenic SOD1G93A and PFN1 ALS mice) and detects subclinical perturbations in net transport, as analyzed in C9orf72 transgenic mice. It also defines a progressive reduction in net transport with aging. To address whether our technique enables drug development, I evaluated therapeutic benefits of (1) gene editing and (2) mutant gene silencing (with RNAi targeting SOD1) in SOD1G93A transgenic mice by characterizing their net axonal transport profiles. I constructed a computational model to evaluate key molecular processes affected in net axonal transport in ALS mouse model. The model allows prediction of key parameters affected in a C9ORF72 BAC transgenic mouse line. Prior immunization with tetanus toxoid does not preclude use of this assay, and it can be used repetitively in the same subject. This assay of net axonal transport offers broad clinical application as a diagnostic tool for motor neuron diseases and as a biomarker for rapid detection of benefit from therapies for transport dysfunction in a range of motor neuron diseases

Exploiting 3D differentiation of human stem cells

"Neurological disorders are a major public health problem and are expected to rise dramatically together with the higher life expectancy and the shift towards an ageing society. Current therapeutic options can only ameliorate some of the symptoms and there are no effective treatments to target pathological mechanisms and stop disease progression. The human brain complexity hampers the understanding of the brain functioning at the molecular, cellular, and pathophysiological levels for many neurological disorders. This highlights the need for new brain models, which can contribute to unveil molecular mechanisms of neurological disorders, identify therapeutic targets and evaluate preclinically new therapies in a more adequate and predictive basis, withstanding its successful translation to the clinics. Despite their undeniable value, traditional animal models diverge from humans at biochemical and genetic levels. Moreover, 2D in vitro cell-based models do not mimic important aspects of brain cellular heterogeneity, architecture and microenvironment (...)".N/

Neurofly 2008 abstracts : the 12th European Drosophila neurobiology conference 6-10 September 2008 Wuerzburg, Germany

This volume consists of a collection of conference abstracts

Mitochondria-endoplasmic reticulum contacts in neuronal cells : from physiology to therapeutics

Mitochondria and the endoplasmic reticulum (ER) are intracellular organelles that play vital physiological functions. Mitochondria are key players in energy production through adenosine triphosphate (ATP) production and calcium (Ca2+) buffering, while the ER is involved in protein and lipid synthesis along with Ca2+ signalling in the cell. In the last 10 years scientists have realised the importance of intracellular organelle communication as a pivotal process for physiological functions. Among these interactions, mitochondria and ER functionally and structurally interact with each other forming mitochondria-ER contact sites (MERCS). Importantly, these structures oversee a variety of pathways including intracellular Ca2+ signalling. Indeed, ER to mitochondria Ca2+ shuttling has been shown to impact on mitochondrial respiration and bioenergetics. On the other hand, sustained increase in Ca2+ signalling between these two organelles can cause activation of apoptosis mediators leading to cell death. In Alzheimer´s disease (AD), cerebral hypometabolism, mitochondrial dysfunction, and functional and structural upregulation of ER to mitochondria apposition appear as early events in disease pathogenesis. Despite over 30 years of studies, the causes of AD are essentially unknown and only two symptomatic drugs have been approved for treatment, which means that AD leads to decline of quality of life and ultimately death. In this thesis, using human brain biopsies from idiopathic normal pressure hydrocephalus (iNPH) patients, mouse models of AD and cellular models, we investigated the role of mitochondria and MERCS in synapses and exocytotic mechanism and their role in the development of pathology in AD. Additionally, we have set up a high throughput screen (HTS) to find potential modulators of mitochondrial function with the overarching aim to find drugs to target neurodegeneration. In PAPER I, for the first time we have shown the presence of several organelle contact sites in human brain material and we have confirmed the presence of MERCS in human synapses. In this study we have also shown that patients suffering from dementia have more MERCS compared to non-demented patients. Furthermore, we have shown correlation of soluble Aβ levels, thought to be one of the initiators of AD, and MERCS number in iNPH patients. In PAPER II, through knockdown of Mitofusin 2 (Mfn2) in SH-SY5Y cells, a negative regulator of MERCS, we have detected substantial increased juxtaposition between ER and mitochondria. Upon Mfn2 knockdown, we have observed decreased levels of cytoplasmic vesicle and increased vesicle release upon cellular depolarization. Furthermore, we have shown that this mechanism was dependent on IP3Rs activity, an important channel for Ca2+ transfer from ER to mitochondria. In PAPER III we have characterised in vitro a novel knock-in model of AD, the AppNL-F model, which overcomes the problem of overexpressing amyloid precursor protein (APP). We have shown that embryonic cells derived from AppNL-F mice are capable of secreting levels of Aβ similar to adult brains, causing bioenergetics impairments, movement abnormalities along neurites and increased MERCS functions. Furthermore, these cells seem to be more susceptible to cell death upon inhibition of mitochondrial respiration compared to WT cells. In PAPER IV, we have assessed whether the other pathological protein in AD, tau, impacts on mitochondrial function and MERCS using the pure tauopathy model P301s. We detected that before tau pathology onset, at 22 days post-natal, animals displayed mitochondrial respiration dysfunctions and increase in MERCS. This pathology was sustained throughout mice life up to 10 months of age. In PAPER V, setting up a HTS platform evaluating mitochondrial enhancers, we have found luteolin, a natural compound from the flavonoid family, to be capable of increasing ATP production in vitro in SH-SY5Y cells and primary cortical neurons, and ex vivo in isolated mitochondria and synaptosomes. The ATP increase shown was due to increased ER to mitochondria juxtaposition and Ca2+ transfer. We have further tested luteolin in Huntington’s disease mutations bearing primary cortical neurons and C.elegans, showing improvement in respiration in vitro and recovery in movement in vivo. In conclusion, this thesis has contributed to expand the knowledge on the role of mitochondria and MERCS in synapses and in exocytotic mechanisms. We have further shown that MERCS and bioenergetics dysfunction occur early during the pathogenic development of disease in tau and amyloid AD models. We have also provided a platform for the study of drugs in neuronal cells, revealing luteolin as a promising enhancer of mitochondrial function