Biology subjects

Rostami, I.

Publications and source records attributed to Rostami, I..

7 recordsLinked to original sources

bioRxiv2025

Advancing Human iPSC-Derived Motor Neuron Models Using Glutamatergic Modulators for Synaptic Function Studies

Understanding human synapse structure and function at nanoscale resolution requires experimental systems that support both high-quality imaging and robust synaptogenesis. While brain tissue and primary neuronal cultures have been widely used, human induced pluripotent stem cells (iPSCs) offer key advantages: they enable patient-specific modeling and provide a renewable source of human neurons without relying on animal models. However, reproducible synapse formation in iPSC-derived neurons, particularly motor neurons (MNs), remains challenging due to variable differentiation efficiencies and low synaptic density. Here, we optimized MN differentiation to enhance synaptogenesis and enable high-resolution structural analysis using cryo-electron tomography (cryo-ET). Within 28 days, human iPSC-derived MNs developed into morphologically and functionally mature neuronal networks, characterized by phase-bright somata, elongated axons, and dense axodendritic branching. To further promote synaptic connectivity, we applied the glutamatergic modulators CX516 and CDPPB during differentiation. Their effects were validated by immunolabeling of synaptic markers, electrophysiology, calcium imaging, live-cell synaptic vesicle recycling assays, and ultrastructural imaging, including cryo-ET. This combined approach of optimized differentiation and targeted neuromodulation yielded reproducible MN networks with enhanced synaptic density and function, providing a robust in vitro platform for investigating human MN physiology, synaptic mechanisms, and disease-relevant synaptopathies.

neuroscience↗

bioRxiv2025

Differentiated SH-SY5Y Cells Exhibit Neuronal Features but Lack Synaptic Maturity

A vital question in neuroscience is if and how efficiently cellular models may be differentiated into functional neuronal cells in culture. Despite the frequent use of the human neuroblastoma cell line SH-SY5Y, differentiation protocols vary extensively, with the most common being differentiation via the addition of retinoic acid and brain-derived neurotrophic factor. However, due to the lack of a reliable evaluation method, their adequacy as synaptic models remains unclear. Here, we investigate whether SH-SY5Y cells constitute a functional model for synaptic studies by phenotypically and ultra structurally analyzing synaptogenesis in SH-SY5Y cells subjected to different differentiation protocols. Electron microscopy (EM) techniques, including conventional EM, cryo-EM, and cryo-electron tomography, were systematically applied to characterize synaptogenesis in SH-SY5Y cells. Further characterization was performed through immunostaining and functional assays, such as live exocytosis assays and whole-cell patch-clamp electrophysiology. Despite exhibiting some presynaptic-like features, differentiated SH-SY5Y cells do not form morphologically or functionally complete synapses under the conditions tested. Immunostaining results were consistent with previous findings, showing synaptic markers. However, functional investigations did not detect synaptic activity. High-throughput EM analyses revealed an absence of synaptic structures in these cells. Additionally, an alternative differentiation approach incorporating additional neurotrophic factors promoted the formation of pre-synaptic-like compartments containing synapse-like synaptic vesicles (SVLVs). Though these SVLVs exhibited pleomorphic size distributions, differing from typical synaptic vesicles, and lacked connectors. These findings emphasize the need for cautious interpretation of results derived from SH-SY5Y cells when studying molecular synaptic architecture or neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo

BackgroundAbnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinsons disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. MethodsFluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively. ResultsTHK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinsons disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. ConclusionWe demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

neuroscience↗

bioRxiv2023

Post-fibrillization nitration of alpha-synuclein abolishes its seeding activity and pathology formation in primary neurons and in vivo

Increasing evidence points to post-translational modifications (PTMs) as key regulators of alpha-synuclein (-Syn) function in health and disease. However, whether these PTMs occur before or after -Syn pathology formation and their role in regulating -Syn toxicity remain unclear. In this study, we demonstrate that post-fibrillization nitration of -Syn fibrils induced their fragmentation, modified their surface and dynamic properties but not their structure, and nearly abolished their seeding activity in primary neurons and in vivo. Furthermore, we show that the dynamic and surface properties of the fibrils, rather than simply their length, are important determinants of -Syn fibril seeding activity. Altogether, our work demonstrates that post-aggregation modifications of -Syn may provide novel approaches to target a central process that contributes to pathology formation and disease progression. Finally, our results suggest that the pattern of PTMs on pathological aggregates, rather than simply their presence, could be a key determinant of their toxicity and neurodegeneration. This calls for reconsidering current approaches relying solely on quantifying and correlating the level of pathology to assess the efficacy of novel therapies, as not all -Syn aggregates in the brain are pathogenic.

neuroscience↗

bioRxiv2021

A novel mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein fibrils and recapitulates its pathological diversity

A novel mutation (E83Q), the first in the NAC domain of alpha-synuclein (aSyn), was recently identified in a patient with dementia with Lewy bodies. We investigated the effects of this mutation on the aggregation of aSyn monomers and the structure, morphology, dynamic, and seeding activity of the aSyn fibrils in neurons. We found that it dramatically accelerates aSyn fibrillization and results in the formation of fibrils with distinct structural and dynamic properties. In cells, this mutation is associated with higher levels of aSyn, accumulation of pS129, and increased toxicity. In a neuronal seeding model of Lewy bodies (LB) formation, the E83Q mutation significantly enhances the internalization of fibrils into neurons, induce higher seeding activity and results in the formation of diverse aSyn pathologies, including the formation of LB-like inclusions that recapitulate the immunohistochemical and morphological features of brainstem LBs observed in PD patient brains. TeaserA novel mutation (E83Q) exacerbates alpha-synuclein aggregation and toxicity and reproduces PD pathological diversity.

neuroscience↗

bioRxiv2021

A new chemoenzymatic semisynthetic approach provides novel insight into the role of phosphorylation beyond exon1 of Huntingtin and reveals N-terminal fragment length-dependent distinct mechanisms of aggregation

Huntingtons disease is a neurodegenerative disorder caused by the expansion of a polyglutamine (poly Q) repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Htt exon1 (Httex1). Herein, we investigated the aggregation properties of mutant N-terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1. We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded novel insights into how PTMs and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slowed its aggregation, whereases phosphorylation at T107 and S116 accelerated the aggregation of Htt171, underscoring the importance of crosstalk between different PTMs. We demonstrate that mutant Htt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mutant Httex1. These observations suggest that different N-terminal fragments could have distinct mechanisms of aggregation and that a single polyQ-targeting anti-aggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the importance of further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity, pathology formation, and disease progression. Table of content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/436743v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@113aebborg.highwire.dtl.DTLVardef@1349236org.highwire.dtl.DTLVardef@6b273borg.highwire.dtl.DTLVardef@dcad5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Investigating crosstalk among PTMs provides novel insight into the structural basis underlying the differential effects of Nt17 PTMs on mutant Httex1 aggregation

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntingtons disease. Our groups previous studies suggested that the Nt17 PTM code is a combinatorial code that involves a complex interplay between different PTMs. Here, we expand on these studies by investigating the effect of methionine 8 oxidation (oxM8) and crosstalk between this PTM and either lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1. We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mutant Httex1 aggregates. This delay in aggregation kinetics could be attributed to the transient accumulation of oligomeric aggregates, which disappear upon the formation of Httex1 oxM8 fibrils. Interestingly, the presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization, whereas the presence of oxM8 did not influence the aggregation inhibitory effect of pT3. To gain insight into the structural basis underlying these proteins aggregation properties, we investigated the impact of each PTM and the combination of these PTMs on the conformational properties of the Nt17 peptide by circular dichroism spectroscopy and molecular dynamics simulation. These studies show that M8 oxidation decreases the helicity of the Nt17 in the presence or absence of PTMs and provides novel insight into the structural basis underlying the effects of different PTMs on mutant Httex1 aggregation. PTMs that lower the mutant Httex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and phosphorylation at Serine 13) result in stabilization and increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation of mutant Httex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mutant Httex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mutant Httex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mutant Httex1 aggregation. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/432155v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@15dbf84org.highwire.dtl.DTLVardef@1c8a50corg.highwire.dtl.DTLVardef@1cbe0c6org.highwire.dtl.DTLVardef@120e0a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗