Biology subjects

Parish, C.

Publications and source records attributed to Parish, C..

8 recordsLinked to original sources

bioRxiv2025

Spatially precise neuron formation via hydrogel mediated modulation of the host astrocyte response

The regenerative capacity of the central nervous system after injury or disease is limited. Advancements in genetic and epigenetic reprogramming of non-neuronal cells into induced neurons presents a promising strategy for neuronal replacement and circuit reconstruction yet is hindered by suboptimal reprogramming efficacy. Here, we have achieved high astrocyte-to-neuron reprogramming efficacy through the ectopic expression of a SOX2 transcription factor using adeno-associated viral vectors. We subsequently designed a hybrid composite biomaterial to act as an implantable reprogramming workshop that perform sequential operations. Once injected, this bespoke system forms a tissue mimetic hydrogel encouraging the ingress of astrocytes to act as our raw material. We demonstrate that the system localized and enhanced the spatially confined delivery of reprogramming factors, entrapping astrocytes to then pass through an intermediate neuroblast state to robustly yield mature neurons. Sustained delivery of valproic acid within the system further promoted neuronal maturation. Overall, using design rules informed by requirements of the reprogramming strategy, we have designed a smart delivery system optimized for both the target tissue and reprogramming factors in a minimally invasive manner to enhance neural repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/695501v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@397e41org.highwire.dtl.DTLVardef@90047forg.highwire.dtl.DTLVardef@1175831org.highwire.dtl.DTLVardef@19f30eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Igniting full-length isoform analysis in single-cell and spatial RNA-seq data with FLAMESv2

Long-read single-cell RNA-sequencing enables the profiling of RNA isoform expression and alternative splicing at single cell resolution. However, diverse single-cell technologies and sparse isoform data demand flexible and accurate analysis tools. We introduce FLAMESv2, a highly modular and protocol-agnostic R/Bioconductor package for long-read single-cell RNA-seq data analysis. FLAMESv2 supports a wide range of single-cell and spatial protocols, is highly configurable, scales to allow multi-sample analysis and provides versatile visualisation and analysis outputs. We demonstrate its compatibility with both droplet-based and combinatorial barcoding single-cell methods, as well as spatial transcriptomics workflows. Benchmarking confirms FLAMESv2 achieves field-leading performance across key analysis tasks. Applying FLAMESv2 to in vitro differentiation of stem cells into neurons, we identify cell-types, differentiation trajectories, expression of annotated and novel isoforms and isoform expression diversity and heterogeneity within individual cells. FLAMESv2 provides a comprehensive, flexible approach to analysing long-read single-cell RNA-sequencing, unlocking this powerful methodology for RNA isoform characterisation.

bioinformatics↗

bioRxiv2025

Transgenic A53T mice have astrocytic α-synuclein aggregates in dopamine and striatal regions

AimsParkinsons disease is considered biologically a neuronal -synuclein disease, largely ignoring the more widespread -synuclein deposition that occurs in astrocytes, with the aim of this study to identify whether astrocytes accumulate small -synuclein aggregates before or after neurons. MethodsFixed serial midbrain and striatal sections from M83 A53T transgenic mouse model of Parkinsons disease and wild-type controls were histologically processed for multiplex labelling of -synuclein and astrocytic markers and astrocyte quantitation performed on digital images using QuPath software. ResultsThe density of astrocytes within the substantia nigra pars compacta was approximately 30% greater compared with other sampled regions (P<0.005). Small aggregates of -synuclein were observed in astrocytic processes, including in wild-type mice where a quarter of all astrocytes had an obvious -synuclein aggregate. Compared to wild-type, A53T transgenic astrocytes had significantly enlarged somas (P<0.001) with more processes (P<0.001) consistent with a reactive phenotype. The A53T transgenic mice had more than double the numbers of astrocytes (P<0.001) and 2.5 times more astrocytes with -synuclein aggregates compared to wild-type mice (P<0.001). ConclusionsThese data suggest that small -synuclein aggregates are normally cleared by astrocytes and that the substantia nigra pars compacta requires more astrocytic support for this function than other midbrain dopaminergic regions or the striatum. This adds another vulnerability factor to those already known for the substantia nigra with early deficits in clearance of small -synuclein aggregates by astrocytes associated with an increased astrocytic reactivity in the A53T transgenic mouse model. Key PointsO_LIThe substantia nigra pars compacta contains a higher density of astrocytes than the ventral tegmental area or striatum, indicating a greater reliance on astrocytic function and a greater vulnerability to astrocyte dysfunction C_LIO_LISmall aggregates of -synuclein were observed in wild-type midbrain and striatal astrocytes, indicating normal clearance of -synuclein by these astrocytes C_LIO_LIMidbrain and striatal astrocytes from A53T transgenic astrocytes have more than double the number of astrocytes and more astrocytes containing -synuclein aggregates which have a reactive morphological phenotype. C_LI

neuroscience↗

bioRxiv2024

Dopamine and cortical iPSC-derived neurons with different Parkinsonian mutations show variation in lysosomal and mitochondrial dysfunction: implications for protein deposition versus selective cell loss

BackgroundMutations causing Parkinsons disease (PD) give diverse pathological phenotypes whose cellular correlates remain to be determined. For example, those with PRKN loss of function mutations have significantly earlier selective vulnerability of dopamine neurons, those with SNCA mutations have increased alpha-synuclein deposition, while those with LRRK2 mutations have additional deposition of tau. Yet all three mutation types are implicated in mitochondrial and/or lysosomal dysfunction. Direct comparison of cell models with these mutations would clarify the relative cellular dysfunctions associated with these different pathological phenotypes. MethodsAn unbiased high-content imaging platform using orthogonal probes to assess both lysosomal and mitochondrial dysfunction, along with alpha-synuclein and tau protein deposition was established using induced pluripotent stem cell (iPSC) derived cortical and ventral midbrain neurons. Three mutation types, SNCA A53T, LRRK2 R1441G and PRKN loss of function (lof), were selected as exemplars of divergent PD pathological phenotypes and compared to each other, and to control iPSC from subjects without PD. ResultsDifferent PD mutations caused cell type specific dysfunctions, likely to impact on both selective neuronal vulnerability and the pathologies observed in PD. Comparison of dopamine neurons identified that both lysosomal and mitochondrial dysfunction were predominant with PRKN lof mutations, whereas immunofluorescent staining revealed that SNCA A53T and LRRK2 R1441G mutations had increased tau deposition. In contrast, cortical neurons with SNCA and LRRK2 mutations both had mitochondrial and autophagy impairments without protein deposition, with LRRK2 cells additionally showing decreased glucocerebrosidase activity and increased alpha-synuclein phosphorylation. ConclusionsLysosomal and mitochondrial dysfunction are predominant in dopamine neurons with PRKN lof mutations, and may drive the early selective loss of dopamine neurons in PRKN mutation carriers. More subtle cellular abnormalities in the SNCA A53T cell lines are likely to predispose to alpha-synuclein aggregation and tau protein deposition over time. The LRRK2 R1441G may also predispose to tau deposition, but despite substantial lysosomal dysfunction with increased alpha-synuclein phosphorylation, pathological alpha-synuclein accumulations were not observed. Understanding the mechanistic differences in how lysosomal and mitochondrial dysfunction impact on PD pathogenesis in different disease subtypes may be important for therapeutic development.

neuroscience↗

bioRxiv2024

Alpha Synuclein Induced Immune Response Triggers Parkinson's Disease Like Symptoms

Increasing evidence suggests that Parkinsons disease is an autoimmune disorder, with findings of elevated peripheral blood mononuclear cell in patients, and antigenic properties of -synuclein driving both the innate and adaptive immunity. Yet, how the interaction of -synuclein and a specific immune response participates to Parkinsons disease ontogenesis has remained unanswered. Here, we reveal that autoimmune response to an -synuclein antigen underlies Parkinsons disease. We demonstrate that autoimmunity mediated by CD4+T cell activation with -synuclein -syn61-75 antigen is required to lead to immune cell infiltration and localized inflammation in the substantia nigra, triggering dopaminergic cell neurodegeneration and deficits in locomotion and gait kinematics. This study offers the first immune-induced mouse model that recapitulates all features of Parkinsons disease to study the mechanisms triggering disease onset. It provides the basis for temporally tracking symptom development, exploring preventive strategies and prodromal therapeutic interventions in Parkinsons Disease. In briefPeripheral -synuclein immunization causes Parkinsons disease-like symptoms in mice. Highlights- Both CD4+ T cells and -synuclein are essential for Parkinsons disease ontogenesis. - Peripheral injection of -syn61-75 induces significant CD4+ T cell infiltration in the mouse brain. - -syn61-75 immunization is associated with inflammation, -synuclein aggregation and dopaminergic cell loss in the substantia nigra pars compacta. - Levodopa-sensitive motor symptoms are detected 8 weeks following -syn61-75 immunization in mice. - This study offers a novel autoimmune -synuclein induced mouse model of Parkinsons disease.

neuroscience↗

bioRxiv2022

Combined scattering, interferometry and fluorescence oblique illumination for live cell nanoscale imaging

To determine the molecular and/or mechanical basis of cell migration using live cell imaging tools, it is necessary to correlate multiple 3D spatiotemporal events simultaneously. Fluorescence nanoscopy and label free nanoscale imaging can complement each other by providing both molecular specificity and structural dynamics of sub-cellular structure. In doing so, a combined imaging system would permit quantitative 3D spatial temporal detail of individual cellular components. In this paper, we empirically determined a series of optimal azimuthal scanning angles and rotating beam to achieve simultaneous and label-free nanoscale and fluorescence imaging. Label-free nanoscale imaging here refers to interferometric, brightfield (BF) and darkfield (DF) rotating coherence scattering (ROCS) microscopy, while fluorescence refers to high inclined Laminated Oblique (HiLO) and total internal reflection fluorescence (TIRF) imaging. The combined capabilities of interferometric, scattering and fluorescence imaging enables (1) the identification of molecular targets (substrate or organelle), (2) quantification of 3D cell morphodynamics, and (3) tracking of intracellular organelles in 3D. This combined imaging tool was then used to characterize migrating platelets and adherent endothelial cells, both critical to the process of infection and wound healing. The combined imaging results of over [~]1000 platelets, suggested that serum albumin (bovine) was necessary for platelets to migrate and scavenge fibrin/fibrinogen. Furthermore, we determine new asynchronous membrane fluctuations between the leading and rear edge of a migrating platelet. We further demonstrated that interferometric imaging permitted the quantification of mitochondria dynamics on lung microvascular cells (HMVEC). Our data suggests that axial displacement of mitochondria is minimized when it is closer to the nucleus or the leading edge of a cell membrane that exhibits retrograde motion. Taken together, this combined imaging platform has proven to quantify multiple spatial temporal events of a migrating cell, that will undoubtedly open ways to new quantitative correlative nanoscale live cell imaging.

biophysics↗

bioRxiv2019

VIRAL DELIVERY OF GDNF PROMOTES FUNCTIONAL INTEGRATION OF HUMAN STEM CELL GRAFTS IN PARKINSONS DISEASE

The derivation of neurotransmitter and region-specific neuronal populations from human pluripotent stem cells (PSC) provides impetus for advancing cell therapies into the clinic. At the forefront is our ability to generate ventral midbrain (VM) dopaminergic (DA) progenitors, suitable for transplantation in Parkinsons disease (PD). Pre-clinical studies, however, have highlighted the low proportion of DA neurons within these grafts and their inferior plasticity by comparison to human fetal donor transplants. Here we sought to examine whether modification of the host environment, through viral delivery of a developmentally critical molecule, glial cell line-derived neurotrophic factor (GDNF), could improve graft survival, integration and function in Parkinsonian rodents. Utilising LMX1A- and PITX3-GFP hPSC reporter lines, we tracked the response of DA progenitors implanted into either a GDNF-rich environment, or in a second group, after a 3-week delay in onset of exposure. We found that early exposure of the graft to GDNF promoted survival of DA and non-DA cells, leading to enhanced motor recovery in PD rats. Delayed overexpression of intrastriatal GDNF also promoted motor recovery in transplanted rats, through alternate selective mechanisms including enhanced A9/A10 specification, increased DA graft plasticity, greater activation of striatal neurons and elevated DA metabolism. Lastly, transcriptional profiling of the grafts highlighted novel genes underpinning these changes. Collectively these results demonstrate the potential of targeted neurotrophic gene therapy strategies to improve human PSC graft outcomes.

neuroscience↗

bioRxiv2019

Massive parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a novel genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction is quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine variants effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino-acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in 2,398 patients treated with thiopurines, with 100% sensitivity and specificity, in contrast with poor performance of bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,103 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.

genomics↗