Biology subjects

Graef, J. D.

Publications and source records attributed to Graef, J. D..

3 recordsLinked to original sources

bioRxiv2022

Elevated alpha-synuclein attenuates phagocytosis in SNCA triplication human iPSC-derived neuron:microglia co-cultures

Synucleinopathies such as Parkinsons disease (PD) are characterized by pathologic production, aggregation, and cell-to-cell transmission of -synuclein (-syn) protein that results in impaired cellular function. While neurons of the substantia nigra pars compacta express high levels of -synuclein and are highly vulnerable to its aberrant expression or conformation, brain-resident macrophages (microglia) are also sensitive to abnormal -synuclein, with recent reports indicating that elevated levels impair phagocytic ability in vivo and in vitro. To explore the impact of elevated -syn on microglial function we employed a co-culture model containing iPSC-derived neurons and microglia-like cells. iPSCs from healthy control donors and a Parkinsons donor with an allelic triplication of the SNCA gene locus were differentiated into neurons and microglia-like cells. In monoculture, neurons and microglia generated from the SNCA triplication donor expressed higher levels of SNCA transcript and protein. Neurons were found to have significantly greater expression of SNCA compared to microglia, regardless of donor genotype. Co-cultures of neurons and microglia revealed that microglia cultured with SNCA triplication neurons displayed reduction in phagocytosis of fluorescent E. coli, irrespective of microglia donor genotype. SNCA mRNA and protein expression could be reduced with treatment with an antisense oligonucleotide (ASO) targeting SNCA. ASO treatment partially rescued microglia phagocytosis in SNCA triplication co-cultures and in co-cultures containing SNCA triplication neurons and healthy control microglia. Our results complement and extend previous findings of impaired microglial function in the presence of elevated -synuclein in a novel patient-derived co-culture model that utilizes more disease-relevant conditions rather than the relaying on the addition of exogenous -synuclein.

neuroscience↗

bioRxiv2019

Partial FMRP expression is sufficient to normalize neuronal hyperactivity in Fragile X neurons

Fragile X Syndrome (FXS) is the most common genetic form of intellectual disability caused by a CGG repeat expansion in the 5-UTR of the Fragile X mental retardation gene FMR1, triggering epigenetic silencing and the subsequent absence of the protein, FMRP. Reactivation of FMR1 represents an attractive therapeutic strategy targeting the genetic root cause of FXS. However, largely missing in the FXS field is an understanding of how much FMR1 reactivation is required to rescue FMRP-dependent mutant phenotypes. Here, we utilize FXS patient derived excitatory neurons to model FXS in vitro and confirm that the absence of FMRP leads to neuronal hyperactivity. We further determined the levels of FMRP and the percentage of FMRP positive cells necessary to correct this phenotype utilizing a mixed and mosaic neuronal culture system and a combination of CRISPR, antisense and expression technologies to titrate FMRP in FXS and WT neurons. Our data demonstrate that restoration of greater than 5% of overall FMRP expression levels or greater than 20% FMRP expressing neurons in a mosaic pattern is sufficient to normalize a FMRP-dependent, hyperactive phenotype in FXS iPSC-derived neurons.\n\nHighlightsO_LICRISPR gene editing to generate FMRP KO and CGG-deleted isogenic iPSCs\nC_LIO_LIMEA as an approach to identify FMR1 dependent phenotype in NGN2 neurons derived from FXS and FMRP KO iPSCs\nC_LIO_LICell mixing paradigm as mosaicism in a dish to rescue phenotype\nC_LIO_LIMinimal level of FMRP determined by FMR1 mRNA and targeted demethylation of CGG repeats to correct the hyperactive phenotype in FXS neurons\nC_LIO_LIASO titration-validated partial expression of FMRP is sufficient to normalize increased neuronal activity\nC_LI

neuroscience↗

bioRxiv2019

Characterization of pathology-inducing α-synuclein species from human diseased brain tissue

Synucleinopathies are a group of neurodegenerative diseases characterized by the presence of pathological accumulations of misfolded, phosphorylated -synuclein (Syn) protein. Multiple lines of evidence indicate that synucleinopathy disease progression is driven by a prion-like process of transmission of a pathologic form of Syn. One potential therapeutic approach to prevent cell-to-cell propagation is to target this transmissible species with selective antibodies. In this study, a rodent primary neuronal culture reporter system was developed to monitor induction of detergent-insoluble, phosphorylated (pS129) aggregates of Syn. Induction of pS129 Syn pathology was observed with both synthetic Syn fibrils (PFFs) and brain lysates from multiple system atrophy (MSA) patients but not Syn monomers or human brain lysate controls. The induction-competent species in MSA lysates could be enriched by high-speed centrifugation suggesting that it is present as a high molecular weight aggregate. Furthermore, samples derived from brain lysates from Parkinsons disease (PD) and Dementia with Lewy Bodies (DLB) patients also induced pS129 Syn pathology, but required longer incubation times. Lastly, the potential of Syn selective antibodies to immunodeplete induction-competent forms of Syn from both PFF and synucleinopathy brain samples is described. The results demonstrate that antibodies targeting the C-terminal of Syn are most effective for immunodepletion of pathology-inducing forms of Syn from samples derived from human synucleinopathy brains. Furthermore, the data support the hypothesis that antibodies that recognize a C-terminal epitope and exhibit selectivity for oligomeric forms over monomeric forms of Syn represent a desirable target for immunotherapy for synucleinopathy patients.

neuroscience↗