Biology subjects

Legname, G.

Publications and source records attributed to Legname, G..

5 recordsLinked to original sources

bioRxiv2026

PODXL regulates the neuronal entry of α-synuclein aggregates

Cell-to-cell transmission of -synuclein (-Syn) aggregates is a key event in Parkinson's disease (PD) pathogenesis, yet the neuronal surface factors that govern uptake of pathogenic -Syn fibrils remain undefined. We developed a flow cytometry-based CRISPR activation screen for modifiers of -Syn preformed fibril (PFF) uptake by human iPSC-derived cortical neurons. By interrogating 68 genes encoding cell-surface proteins prioritised by PD genetic associations, we identified podocalyxin-like protein 1 (PODXL) as a strong suppressor of -Syn fibril uptake. High-content imaging and PODXL cDNA overexpression confirmed reduced PFF uptake. Stable PODXL overexpression also reduced PFF-seeded pSynSer129 accumulation and uptake of structurally distinct recombinant and disease-derived -Syn fibrils. Conversely, shRNA-mediated PODXL knockdown increased -Syn PFF uptake, and analysis of PODXL domain-deletion and N-glycosylation mutants suggested that the ectodomain and N-glycosylation contribute to the PODXL-mediated reduction in uptake. Transcriptomic profiling further identified membrane metalloendopeptidase (MME) as a PODXL-responsive candidate mediator. MME activation and treatment with conditioned medium from MME-activated neurons reduced PFF uptake. Thiorphan, an MME inhibitor, partially attenuated the PODXL-associated reduction in uptake. Together, our findings identify PODXL as a neuronal brake on -Syn uptake and implicate its glycan-dependent surface properties together with MME-associated extracellular activity.

neuroscience↗

bioRxiv2026

Prion Protein Deficiency Results in Synaptic, Neural Network and Behavioral Alterations

The cellular form of the prion protein (PrPC) is known for its involvement in the pathogenesis of prion diseases. Recent research implicates the physiological isoform of PrP in neuronal development, excitability, and synaptic plasticity, as well as in other biological processes. However, its precise function in the development and function of neurons remains poorly understood. Here, we investigated its role during different developmental stages, both in vitro and in vivo, using different PrP knock-out (KO) mouse lines (Prnp-/-). Prion protein KO neurons cultured on microelectrode arrays (MEAs) displayed altered network dynamics compared to wild type cultures, comprising reduced burst frequency, and abnormal spike patterns, indicative of impaired maturation of the synaptic circuitry. These functional alterations were associated with a reduced expression of key presynaptic and postsynaptic proteins, including elements of the SNARE complex and regulators of excitation-inhibition balance. Similar molecular changes were also confirmed in a second Prnp-/- model, suggesting that PrPC is directly involved in these mechanisms regardless of genetic backgrounds. Alterations in neuronal networks were traceable into adulthood: in vivo recordings in adult Prnp-/- mice revealed increased neuronal responses to visual danger stimuli, which correlated with behaviorally increased fear responses to those stimuli. Together, our findings support a critical role for PrPC in the establishment and maintenance of functional neuronal networks, from early developmental stages in vitro to behaviorally mature relevant circuits in vivo, beyond genomic background. These results indicate that PrPC acts as a key regulator of synaptic development and function both in physiological and pathological conditions.

molecular biology↗

bioRxiv2021

Neuronal hemoglobin induces α-synuclein cleavage and loss of dopaminergic neurons

BackgroudParkinsons disease (PD) presents the selective loss of A9 dopaminergic (DA) neurons of Substantia Nigra pars compacta (SNpc) and the presence of intracellular aggregates called Lewy bodies. -synuclein (-syn) species truncated at the carboxy terminal (C-terminal) accumulate in pathological inclusions and promote -syn aggregation and toxicity. Hemoglobin (Hb) is the major oxygen carrier protein in erythrocytes. In addition, Hb is expressed in A9 DA neurons where it influences mitochondrial activity. Hb overexpression increases cells vulnerability in a neurochemical model of PD in vitro and forms cytoplasmic and nucleolar aggregates upon short-term overexpression in mouse SNpc. Methods and {beta}-globin chains were co-expressed in DA cells of SNpc in vivo upon stereotaxic injections of an Adeno-Associated Virus isotype 9 (AAV9) and in DA iMN9D cells in vitro. ResultsLong-term Hb over-expression in SNpc induced the loss of about 50% of DA neurons, a mild motor impairment and deficits in recognition and spatial working memory. Hb triggered the formation of endogenous -synuclein C-terminal truncated species. Similar -syn fragments were found in vitro in DA iMN9D cells over-expressing and {beta}-globins when treated with pre-formed -syn fibrils. ConclusionOur study positions Hb as a relevant player in PD pathogenesis for its ability to trigger DA cells loss in vivo and the formation of C-terminal -synuclein fragments.

neuroscience↗

bioRxiv2020

The uptake of tau amyloid fibrils is facilitated by the cellular prion protein and hampers prion propagation in cultured cells

Tauopathies are prevalent, invariably fatal brain diseases for which no cure is available. Tauopathies progressively affect the brain through cell-to-cell transfer of tau protein amyloids, yet the spreading mechanisms are unknown. Here we show that the cellular prion protein (PrPC) facilitates the uptake of tau aggregates by cultured cells, possibly by acting as an endocytic receptor. In mouse neuroblastoma cells, we found that tau amyloids bind to PrPC; internalization of tau fibrils was reduced in isogenic cells devoid of the gene encoding PrPC. Antibodies against N-proximal epitopes of PrPC impaired the binding of tau amyloids and decreased their uptake. Surprisingly, exposure of chronically prion-infected cells to tau amyloids reduced the accumulation of aggregated prion protein; this effect lasted for more than 72 hours after amyloid removal. These results point to bidirectional interactions between the two proteins: whilst PrPC mediates the entrance of tau fibrils in cells, PrPSc buildup is greatly reduced in their presence, possibly because of an impairment in the prion conversion process.

neuroscience↗

bioRxiv2018

Deciphering copper coordination in the animal prion protein amyloidogenic domain

Prions are pathological isoforms of the cellular prion protein (PrPC) responsible for transmissible spongiform encephalopathies (TSE). PrPC interacts with copper through unique octarepeat and non-octarepeat (non-OR) binding sites. Previous works on human PrPC suggest that copper binding to the non-OR region may have a role during prion conversion. The molecular details of copper coordination within the non-OR region are not well characterized. By means of small angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its coordination geometries when bound to the non-OR region of recombinant PrPC (recPrP) from animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine PrPC carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); while as highly TSE-susceptible PrPC models we employed OvPrP with polymorphism V136, R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) affects the structural plasticity of the non-OR region leading to a more compacted conformation of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water molecule, making the non-OR region more flexible and open to the solvent. These changes in copper coordination in prion resistant and susceptible species provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.

biochemistry↗