Dataset Information

Multiparametric rapid screening of neuronal process pathology for drug target identification in HSP patient-specific neurons.

ABSTRACT: Axonal degeneration is a key pathology of neurodegenerative diseases, including hereditary spastic paraplegia (HSP), a disorder characterized by spasticity in the lower limbs. Treatments for HSP and other neurodegenerative diseases are mainly symptomatic. While iPSC-derived neurons are valuable for drug discovery and target identification, these applications require robust differentiation paradigms and rapid phenotypic read-outs ranging between hours and a few days. Using spastic paraplegia type 4 (SPG4, the most frequent HSP subtype) as an exemplar, we here present three rapid phenotypic assays for uncovering neuronal process pathologies in iPSC-derived glutamatergic cortical neurons. Specifically, these assays detected a 51% reduction in neurite outgrowth and a 60% increase in growth cone area already 24?hours after plating; axonal swellings, a hallmark of HSP pathology, was discernible after only 5 days. Remarkably, the identified phenotypes were neuron subtype-specific and not detectable in SPG4-derived GABAergic forebrain neurons. We transferred all three phenotypic assays to a 96-well setup, applied small molecules and found that a liver X receptor (LXR) agonist rescued all three phenotypes in HSP neurons, providing a potential drug target for HSP treatment. We expect this multiparametric and rapid phenotyping approach to accelerate development of therapeutic compounds for HSP and other neurodegenerative diseases.

SUBMITTER: Rehbach K

PROVIDER: S-EPMC6610147 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Multiparametric rapid screening of neuronal process pathology for drug target identification in HSP patient-specific neurons.

Rehbach Kristina K Kesavan Jaideep J Hauser Stefan S Ritzenhofen Swetlana S Jungverdorben Johannes J Schüle Rebecca R Schöls Ludger L Peitz Michael M Brüstle Oliver O

Scientific reports 20190703 1

Axonal degeneration is a key pathology of neurodegenerative diseases, including hereditary spastic paraplegia (HSP), a disorder characterized by spasticity in the lower limbs. Treatments for HSP and other neurodegenerative diseases are mainly symptomatic. While iPSC-derived neurons are valuable for drug discovery and target identification, these applications require robust differentiation paradigms and rapid phenotypic read-outs ranging between hours and a few days. Using spastic paraplegia type ...[more]

PMID: 31270336

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Project description:BACKGROUND: LEF1/TCF transcription factors and their activator ?-catenin are effectors of the canonical Wnt pathway. Although Wnt/?-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by ?-catenin in neurons. We recently showed that ?-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new ?-catenin targets in the adult thalamus. RESULTS: We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear ?-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between ?-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of ?-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca(2+)-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by ?-catenin, although the binding of ?-catenin to the regulatory sequences of these genes could not be confirmed. CONCLUSIONS: In the thalamus, ?-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of ?-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies.

Project description:Protein micropatterning is a powerful tool for studying the effects of extracellular signals on cell development and regeneration. Laser micropatterning of proteins is the most flexible method for patterning many different geometries, protein densities, and concentration gradients. Despite these advantages, laser micropatterning remains prohibitively slow for most applications. Here, we take advantage of the rapid multi-photon induced photobleaching of fluorophores to generate sub-micron resolution patterns of full-length proteins on polymer monolayers, with sub-microsecond exposure times, i.e. one to five orders of magnitude faster than all previous laser micropatterning methods. We screened a range of different PEG monolayer coupling chemistries, chain-lengths and functional caps, and found that long-chain acrylated PEG monolayers are effective at resisting non-specific protein adhesion, while permitting efficient cross-linking of biotin-4-fluorescein to the PEG monolayers upon exposure to femtosecond laser pulses. We find evidence that the dominant photopatterning chemistry switches from a two-photon process to three- and four-photon absorption processes as the laser intensity increases, generating increasingly volatile excited triplet-state fluorophores, leading to faster patterning. Using this technology, we were able to generate over a hundred thousand protein patterns with varying geometries and protein densities to direct the polarization of hippocampal neurons with single-cell precision. We found that certain arrays of patterned triangles as small as neurite growth cones can direct polarization by impeding the elongation of reverse-projecting neurites, while permitting elongation of forward-projecting neurites. The ability to rapidly generate and screen such protein micropatterns can enable discovery of conditions necessary to create in vitro neural networks with single-neuron precision for basic discovery, drug screening, as well as for tissue scaffolding in therapeutics.

Dataset Information

Multiparametric rapid screening of neuronal process pathology for drug target identification in HSP patient-specific neurons.

Publications

Multiparametric rapid screening of neuronal process pathology for drug target identification in HSP patient-specific neurons.

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