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Kirik, D.

Publications and source records attributed to Kirik, D..

10 recordsLinked to original sources

Machine learning-based image analysis of Parkinson's disease iPS-derived neurons predicts genotype and reveals mitochondria-lysosome abnormalities

Mitochondrial and lysosomal dysfunction are central features of Parkinsons disease (PD) across major genetic forms including PRKN, SNCA, and LRRK2. We applied cell morphomics, a machine-learning-based framework combining high-content imaging with quantitative feature extraction, to analyse mitochondrial and lysosomal morphology at single-cell resolution in iPS cell-derived cortical neurons from PD patients and healthy controls (13 lines total). Supervised machine-learning models distinguished PD neurons from controls with high accuracy (AUC = 0.87) and reliably separated individual genotypes. Feature importance and attribution analysis revealed genotype-specific organelle biases, with mitochondrial features dominating classification in PRKN neurons, balanced mitochondrial and lysosomal contributions in SNCA neurons, and a greater lysosomal contribution in LRRK2 neurons. Multi-class models retained strong performance, and findings were reproduced across two independent laboratories using different dyes and imaging conditions. These results demonstrate that morphomics provides a robust and scalable framework to quantify genotype-specific organelle abnormalities in PD neurons and supports its application for cellular stratification and biomarker discovery.

neuroscience↗

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↗

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↗

Novel tools to quantify total, phospho-Ser129 and aggregated alpha-synuclein in the mouse brain

Assays for quantifying aggregated and phosphorylated (S129) human -synuclein protein are widely used to evaluate pathological burden in patients suffering from synucleinopathy disorders. Many of these assays, however, do not cross-react with mouse -synuclein or exhibit poor sensitivity for this target, which is problematic considering the preponderance of mouse models at the forefront of pre-clinical -synuclein research. In this project, we addressed this unmet need by reformulating two existing AlphaLISA(R) SureFire(R) UltraTM total and pS129 -synuclein assay kits to yield robust and ultrasensitive (LLoQ [&le;]0.5pg/mL) quantification of mouse and human wild-type and pS129 -synuclein protein. We then employed these assays, together with the BioLegend -synuclein aggregate ELISA, to assess the relationship between -synuclein S129 phosphorylation and aggregation in different mouse brain tissue preparations. Overall, we highlight the compatibility of these new immunoassays with rodent models and demonstrate their potential to advance knowledge surrounding -synuclein phosphorylation and aggregation in synucleinopathies.

neuroscience↗

Robust nuclease-dead S. aureus dCas9-mediated alpha-synuclein knockdown in substantia nigra in a humanized mouse model of Parkinson's disease

Parkinsons disease (PD) is becoming increasingly prevalent due to an aging society, which places a substantial disease burden on patients and their families and an annual cost estimated at 52 billion dollars. However, no approved disease modulatory therapies that halt disease progression are available. Alpha-synuclein is a critical therapeutic target found in aggregated form in Lewy bodies which is the diagnostic hallmark of PD. Familial autosomal dominant forms of PD can present with causative exonic point mutations, copy number multiplications, and non-coding risk variants in the alpha-synuclein gene. The disease onset, severity, and progression depend on the gene expression levels of the alpha-synuclein. Here, we demonstrate that Streptococcus aureus dCas9 (sadCas9)-mediated CRISPR interference (CRISPRi) reduces alpha-synuclein mRNA and protein levels in a humanized mouse model. The mechanism of action is based on the principle that a complementary single guide RNA (sgRNA) recruits the sadCas9 protein to the promoter region of alpha-synuclein and modulates target gene transcription, leading to reduced gene expression. We show robust downregulation of alpha-synuclein in neurons after unilateral stereotactic injection into the substantia nigra of adult mice 1 and 6 months after surgery. This work shows proof of concept that viral-mediated sadCas9 CRISPR interference can be a promising therapeutic strategy to reduce alpha-synuclein in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/556425v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4d9247org.highwire.dtl.DTLVardef@beb419org.highwire.dtl.DTLVardef@21e0f9org.highwire.dtl.DTLVardef@391d5a_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisThis study focuses on the development of an AAV9 nuclease-dead S. aureus CRISPR/Cas9 expression system designed to target the human SNCA promoter. The objective is to achieve downregulation of -synuclein (a-syn) expression. After performing surgery and introducing the expression system, the levels of a-syn were measured at 1 month and 6 months post-surgery. The results indicate a significant downregulation of a-syn at both time points. Furthermore, it was observed that the initial immune response to the system attenuated over time, reaching control levels at the 6-month mark. These findings suggest the potential of this expression system for long-term downregulation of a-syn and provide insights into the immune response dynamics associated with its use.

neuroscience↗

Nuclease-dead S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson's disease

Parkinsons disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies.

neuroscience↗

Combining CRISPR/Cas9 and brain imaging: from genes to proteins to networks

Receptors, transporters and ion channels are important targets for therapy development in neurological diseases including Alzheime[r]s disease, Parkinso[n]s disease, epilepsy, schizophrenia and major depression. Several receptors and ion channels identified by next generation sequencing may be involved in disease initiation and progression but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on whole brain level. Here, we combine CRISPR/Cas9 gene-editing with in vivo positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene networks relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR/Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a novel tool to investigate the impact of specific genes on brain molecular and functional dynamics which will help to develop tailored therapies for normalizing brain function. The method can easily be transferred to higher-order species allowing for a direct comparison of the molecular imaging findings.

neuroscience↗

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↗

Two C-terminal sequence variations determine differential neurotoxicity between human and mouse α-synuclein

-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinsons disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models as well as their biochemical properties relevant to aSyn pathobiology. We report that mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.

neuroscience↗

Organotypic slice culture model demonstrates interneuronal spreading of alpha-synuclein aggregates

Here we describe the use of an organotypic hippocampal slice model for studying -synuclein aggregation and inter-neuronal spreading initiated by injection of preformed -synuclein filaments (PFFs). PFF injection at dentate gyrus templates the endogenous -synuclein to form aggregates in axons and cell bodies that spread to CA3 and CA1 regions. Aggregates were insoluble and phosphorylated at serine 129, recapitulating Lewy pathology features found in Parkinsons disease and other synucleinopathies. The spreading of the aggregates were favoring the anterograde direction in the slice model. The model allowed development of slices expressing only serine-129 phosphorylation-deficient human -synuclein (S129G) using adeno-associated viral (AAV) vector in -synuclein knockout slices. Processes of aggregation and spreading of -synuclein were thereby shown to be independent of phosphorylation at serine 129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated by using viral vectors allow testing of hypotheses on mechanisms involved in formation of -synuclein aggregates and their prion-like spreading.

neuroscience↗