Search

Assays for quantifying aggregated and phosphorylated (S129) human α-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 α-synuclein or exhibit poor sensitivity for this target, which is problematic considering the preponderance of mouse models at the forefront of pre-clinical α-synuclein research. In this project, we addressed this unmet need by reformulating two existing AlphaLISA ® SureFire ® Ultra™ total and pS129 α-synuclein assay kits to yield robust and ultrasensitive (LLoQ ≤ 0.5 pg/mL) quantification of mouse and human wild-type and pS129 α-synuclein protein. We then employed these assays, together with the BioLegend α-synuclein aggregate ELISA, to assess α-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 α-synuclein phosphorylation and aggregation in synucleinopathies., (© 2024. The Author(s).)

Dopaminergic neurons in the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNpc) comprise around 75% of all dopaminergic neurons in the human brain. While both groups of dopaminergic neurons are in close proximity in the midbrain and partially overlap, development, function, and impairments in these two classes of neurons are highly diverse. The molecular and cellular mechanisms underlying these differences are not yet fully understood, but research over the past decade has highlighted the need to differentiate between these two classes of dopaminergic neurons during their development and in the mature brain. This differentiation is crucial not only for understanding fundamental circuitry formation in the brain but also for developing therapies targeted to specific dopaminergic neuron classes without affecting others. In this review, we summarize the state of the art in our understanding of the differences between the dopaminergic neurons of the VTA and the SNpc, such as anatomy, structure, morphology, output and input, electrophysiology, development, and disorders, and discuss the current technologies and methods available for studying these two classes of dopaminergic neurons, highlighting their advantages, limitations, and the necessary improvements required to achieve more-precise therapeutic interventions.

In this article, we propose a post-processing scheme for the novel volumetric microscopy technique SILMAS. We demonstrate this scheme on data from an alpha-synuclein transgenic mouse brain. By combining structured illumination and axial sweeping, a SILMAS measurement provides a prerequisite for quantitative data extraction through improved contrast and optical sectioning. However, due to the technique's efficient removal ofb multiple scattered light, image artifacts such as illumination inhomogeneity, shadowing stripes, and signal attenuation, are highlighted in the recorded volumes. To suppress these artifacts, we rely on the strengths of the imaging method. The SILMAS data, together with the Beer-Lambert law, allow for an approximation of real light extinction, which can be used to compensate for light attenuation in a near-quantitative way. Shadowing stripes can be suppressed efficiently using a computational strategy thanks to the large numerical aperture of an axially swept light sheet. Here, we build upon prior research that employed wavelet-Fourier filtering by incorporating an extra bandpass step. This allows us to filter high-contrast light sheet microscopy data without introducing new artifacts and with minimal distortion of the data. The combined technique is suitable for imaging cleared tissue samples of up to a centimeter scale with an isotropic resolution of a few microns. The combination of a thin and uniform light sheet, scattered light suppression, light attenuation compensation, and shadowing suppression produces volumetric data that is seamless and highly uniform., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s).)

The ubiquitin kinase-ligase pair PINK1-PRKN recognizes and transiently labels damaged mitochondria with ubiquitin phosphorylated at Ser65 (p-S65-Ub) to mediate their selective degradation (mitophagy). Complete loss of PINK1 or PRKN function unequivocally leads to early-onset Parkinson disease, but it is debated whether impairments in mitophagy contribute to disease later in life. While the pathway has been extensively studied in cell culture upon acute and massive mitochondrial stress, basal levels of activation under endogenous conditions and especially in vivo in the brain remain undetermined. Using rodent samples, patient-derived cells, and isogenic neurons, we here identified age-dependent, brain region-, and cell type-specific effects and determined expression levels and extent of basal and maximal activation of PINK1 and PRKN. Our work highlights the importance of defining critical risk and therapeutically relevant levels of PINK1-PRKN signaling which will further improve diagnosis and prognosis and will lead to better stratification of patients for future clinical trials.

Parkinson's 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., (© 2023. Springer Nature Limited.)

High-throughput imaging allows in vitro assessment of neuron morphology for screening populations under developmental, homeostatic, and/or disease conditions. Here, we present a protocol to differentiate cryopreserved human cortical neuronal progenitors into mature cortical neurons for high-throughput imaging analysis. We describe the use of a notch signaling inhibitor to generate homogeneous neuronal populations at densities amenable to individual neurite identification. We detail neurite morphology assessment via measuring multiple parameters including neurite length, branches, roots, segments and extremities, and neuron maturation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Early non-motor features including anxiety, depression and altered social cognition are present in Huntington's disease (HD). The underlying neurobiological mechanisms are not known. Oxytocin (OXT) is involved in the regulation of emotion, social cognition and metabolism, and our previous work showed that the OXT system is affected early in HD. The aim of the study was to investigate the potential causal relationship between the selective expression of mutant huntingtin (mHTT) in OXT neurons and the development of non-motor features and neuropathology., Methods: To express mHTT only in OXT neurons, we used a novel flex-switch adeno-associated viral vector design to selectively express either mHTT or wild-type HTT in the paraventricular nucleus of the hypothalamus using OXT-Cre-recombinase mice. We also performed a mirror experiment to selectively delete mHTT in OXT neurons using the BACHD mouse model. Mice underwent a battery of behavioural tests to assess psychiatric and social behaviours 3 months post-injection or at 2 months of age, respectively. Post-mortem analyses were performed to assess the effects on the OXT system., Results: Our results show that selective expression of mHTT in OXT neurons was associated with the formation of mHTT inclusions and a 26% reduction of OXT-immunopositive neurons as well as increased anxiety-like behaviours compared with uninjected mice. However, selective deletion of mHTT from OXT neurons alone was not sufficient to alter the metabolic and psychiatric phenotype of the BACHD mice at this early time point., Conclusions: Our results indicate that mHTT expression can exert cell-autonomous toxic effects on OXT neurons without affecting the non-motor phenotype at early time points in mice., (© 2023 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Parkinson's disease (PD) is a chronic, neurodegenerative condition characterized by motor symptoms such as bradykinesia, rigidity, and tremor, alongside multiple nonmotor symptoms. The appearance of motor symptoms is linked to progressive dopaminergic neuron loss within the substantia nigra. PD incidence increases sharply with age, suggesting a strong association between mechanisms driving biological aging and the development and progression of PD. However, the role of aging in the pathogenesis of PD remains understudied. Numerous models of PD, including cell models, toxin-induced models, and genetic models in rodents and nonhuman primates (NHPs), reproduce different aspects of PD, but preclinical studies of PD rarely incorporate age as a factor. Studies using patient neurons derived from stem cells via reprogramming methods retain some aging features, but their characterization, particularly of aging markers and reproducibility of neuron type, is suboptimal. Investigation of age-related changes in PD using animal models indicates an association, but this is likely in conjunction with other disease drivers. The biggest barrier to drawing firm conclusions is that each model lacks full characterization and appropriate time-course assessments. There is a need to systematically investigate whether aging increases the susceptibility of mouse, rat, and NHP models to develop PD and understand the role of cell models. We propose that a significant investment in time and resources, together with the coordination and sharing of resources, knowledge, and data, is required to accelerate progress in understanding the role of biological aging in PD development and improve the reliability of models to test interventions., Competing Interests: Conflict of Interest statement All authors declare that they have no competing interests.

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-syn's membrane binding region. This allows enhancement of the "double-anchor" vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation, and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from α-syn aggregate-related diseases., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

Receptors, transporters, and ion channels are important targets for therapy development in neurological diseases, 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 the whole-brain level. We combine CRISPR-Cas9 gene editing with in vivo positron emission tomography (PET) and functional MRI (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 network 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 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.

Light-sheet fluorescent microscopy (LSFM) has, in recent years, allowed for rapid 3D-imaging of cleared biomedical samples at larger and larger scale. However, even in cleared samples, multiple light scattering often degrades the imaging contrast and widens the optical sectioning. Accumulation of scattering intensifies these negative effects as light propagates inside the tissue, which accentuates the issues when imaging large samples. With axially swept light-sheet microscopy (ASLM), centimeter-scale samples can be scanned with a uniform micrometric optical sectioning. But to fully utilize these benefits for 3D-imaging in biomedical tissue samples, suppression of scattered light is needed. Here, we address this by merging ASLM with light-sheet based structured illumination into Structured Illumination Light-sheet Microscopy with Axial Sweeping (SILMAS). The SILMAS method thus enables high-contrast imaging, isotropic micrometric resolution and uniform optical sectioning in centimeter-scale scattering samples, creating isotropic 3D-volumes of e.g., whole mouse brains without the need for any computation-heavy post-processing. We demonstrate the effectiveness of the approach in agarose gel phantoms with fluorescent beads, and in an PFF injected alpha-synuclein transgenic mouse model tagged with a green fluorescent protein (SynGFP). SILMAS imaging is compared to standard ASLM imaging on the same samples and using the same optical setup, and is shown to increase contrast by as much as 370% and reduce widening of optical sectioning by 74%. With these results, we show that SILMAS improves upon the performance of current state-of-the-art light-sheet microscopes for large and imperfectly cleared tissue samples and is a valuable addition to the LSFM family., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson's disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)