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Aims: Metabolic dysfunction is involved in modulating the disease process in Huntington disease (HD) but the underlying mechanisms are not known. The aim of this study was to investigate if the metabolic regulators sirtuins are affected in HD. Methods: Quantitative real‐time polymerase chain reactions were used to assess levels of SIRT1‐3 and downstream targets in post mortem brain tissue from HD patients and control cases as well as after selective hypothalamic expression of mutant huntingtin (HTT) using recombinant adeno‐associated viral vectors in mice. Results: We show that mRNA levels of the metabolic regulator SIRT1 are increased in the striatum and the cerebral cortex but not in the less affected cerebellum in post mortem HD brains. Levels of SIRT2 are only increased in the striatum and SIRT3 is not affected in HD. Interestingly, mRNA levels of SIRT1 are selectively increased in the lateral hypothalamic area (LHA) and ventromedial hypothalamus (VMH) in HD. Further analyses of the LHA and VMH confirmed pathological changes in these regions including effects on SIRT1 downstream targets and reduced mRNA levels of orexin (hypocretin), prodynorphin and melanin‐concentrating hormone (MCH) in the LHA and of brain‐derived neurotrophic factor (BDNF) in the VMH. Analyses after selective hypothalamic expression of mutant HTT suggest that effects on BDNF, orexin, dynorphin and MCH are early and direct, whereas changes in SIRT1 require more widespread expression of mutant HTT. Conclusions: We show that SIRT1 expression is increased in HD‐affected brain regions and that metabolic pathways are altered in the HD hypothalamus. [ABSTRACT FROM AUTHOR]

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.)

Background: α-synuclein (α-syn) aggregation can lead to degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) as invariably observed in patients with Parkinson's Disease (PD). The co-chaperone DNAJB6 has previously been found to be expressed at higher levels in PD patients than in control subjects and was also found in Lewy bodies. Our previous experiments showed that knock out of DNAJB6 induced α-syn aggregation in cellular level. However, effects of overexpression of DNAJB6 against α-syn aggregation remains to be investigated., Methods: We used a α-syn CFP/YFP HEK293 FRET cell line to investigate the effects of overexpression of DNAJB6 in cellular level. α-syn aggregation was induced by transfection α-syn preformed fibrils (PPF), then was measured FRET analysis. We proceeded to investigate if DNAJB6b can impair α-syn aggregation and toxicity in an animal model and used adeno associated vira (AAV6) designed to overexpress of human wt α-syn, GFP-DNAJB6 or GFP in rats. These vectors were injected into the SNpc of the rats, unilaterally. Rats injected with vira to express α-syn along with GFP in the SNpc where compared to rats expressing α-syn and GFP-DNAJB6. We evaluated motor functions, dopaminergic cell death, and axonal degeneration in striatum., Results: We show that DNAJB6 prevent α-syn aggregation induced by α-syn PFF's, in a cell culture model. In addition, we observed α-syn overexpression caused dopaminergic cell death and that this was strongly reduced by co-expression of DNAJB6b. The lesion caused by α-syn overexpression resulted in behavior deficits, which increased over time as seen in stepping test, which was rescued by co-expression of DNAJB6b., Conclusion: We here demonstrate for the first time that DNAJB6 is a strong suppressor of α-syn aggregation in cells and in animals and that this results in a suppression of dopaminergic cell death and PD related motor deficits in an animal model of PD., (Copyright © 2021. Published by Elsevier Inc.)

Background: Pathology in the noradrenergic A6 locus coeruleus has not been compared with more rostral dopaminergic A9 substantia nigra and A10 ventral tegmental area, and cholinergic Ch4 basal nucleus and Ch1/2 septal regions in the same cases of Parkinson's disease (PD)., Objective: To determine whether there is a gradient of caudal to rostral cell loss in PD., Methods: Postmortem brains were collected from longitudinally followed donors with PD (n = 14) and aged-matched healthy donors (n = 13), six with restricted brainstem Lewy pathology (RLP), fixed in formalin and serial tissue slabs processed for cell and pathological quantitation. Noradrenergic A6 neurons were assessed and compared with previously published midbrain and basal forebrain data. From these data, regression estimates of pathological onset and progression were determined., Results: Restricted Lewy pathology (RLP) cases had high pathological variability but no significant reduction in neurons. Pathology containing A6 neuron loss started at PD diagnosis and progressed faster (2.4% p.a) than the loss of dopaminergic A9 neurons (2% loss p.a.). Cases with dementia had significantly more pathology in noradrenergic and cholinergic neurons, had greater noradrenergic A6 neuron loss (29% more, progressing at 3.2% p.a.), and a selective loss of lateral A10 nonmelanized dopamine-producing neurons (starting a decade following diagnosis)., Conclusions: These findings show that in the same Parkinson's disease cases cell loss in these neurotransmitter systems does not follow a strict caudal to rostral trajectory and suggests symptom onset may relate to substantial pathology in the noradrenergic A6 locus coeruleus neurons in people with reduced dopamine-producing A9 substantia nigra neurons. © 2021 International Parkinson and Movement Disorder Society., (© 2021 International Parkinson and Movement Disorder Society.)

Aims: Huntington disease (HD) is a fatal neurodegenerative disorder with no disease-modifying treatments approved so far. Ongoing clinical trials are attempting to reduce huntingtin (HTT) expression in the central nervous system (CNS) using different strategies. Yet, the distribution and timing of HTT-lowering therapies required for a beneficial clinical effect is less clear. Here, we investigated whether HD-related behaviours could be prevented by inactivating mutant HTT at different disease stages and to varying degrees in an experimental model., Methods: We generated mutant BACHD mice with either a widespread or circuit-specific inactivation of mutant HTT by using Cre recombinase (Cre) under the nestin promoter or the adenosine A 2A receptor promoter respectively. We also simulated a clinical gene therapy scenario with allele-specific HTT targeting by injections of recombinant adeno-associated viral (rAAV) vectors expressing Cre into the striatum of adult BACHD mice. All mice were assessed using behavioural tests to investigate motor, metabolic and psychiatric outcome measures at 4-6 months of age., Results: While motor deficits, body weight changes, anxiety and depressive-like behaviours are present in BACHD mice, early widespread CNS inactivation during development significantly improves rotarod performance, body weight changes and depressive-like behaviour. However, conditional circuit-wide mutant HTT deletion from the indirect striatal pathway during development and focal striatal-specific deletion in adulthood failed to rescue any of the HD-related behaviours., Conclusions: Our results indicate that widespread targeting and the timing of interventions aimed at reducing mutant HTT are important factors to consider when developing disease-modifying therapies for HD., (© 2020 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

An increase in dopamine (DA) synthesis capacity in the dorsal striatum (DS) during the prodromal stage of schizophrenia becomes more pronounced as patients progress to the full disorder. Understanding this progression is critical to intervening in disease course. We developed an animal model-Enhanced Dopamine in Prodromal Schizophrenia (EDiPS)-which uses a genetic construct to increase DA synthesis capacity in the DS of male rats. We assessed pre-pulse inhibition (PPI) and amphetamine (AMPH)-induced locomotion (0.6 mg/kg) in EDiPS animals longitudinally after post-natal day 35 (when the EDiPS construct is administered). We also assessed their response to repeated acute restraint stress. In adult EDiPS animals, we measured baseline and evoked extracellular DA levels, and their stereotyped responses to 5 mg/kg AMPH. AMPH-induced hyperlocomotion was apparent in EDiPS animals 6-weeks after construct administration. There was an overall PPI deficit in EDiPS animals across all timepoints, however the stress response of EDiPS animals was unaltered. Adult EDiPS animals show normal baseline and potassium-evoked DA release in the DS. These findings suggest that key behavioural phenotypes in EDiPS animals show a progressive onset, similar to that demonstrated by patients as they transition to schizophrenia. The EDiPS model could therefore be used to investigate the molecular mechanisms underlying the prodrome of schizophrenia.

Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histological analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA., (Copyright © 2020. Published by Elsevier Inc.)

Background: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson's 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., Methods: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey's multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn's multiple comparisons test or a two-tailed Mann-Whitney test., Results: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology., Conclusions: 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.

Dopaminergic neurons (DAns), generated from human pluripotent stem cells (hPSCs), are capable of functionally integrating following transplantation and have recently advanced to clinical trials for Parkinson's disease (PD). However, pre-clinical studies have highlighted the low proportion of DAns within hPSC-derived grafts and their inferior plasticity compared to fetal tissue. Here, we examined whether delivery of a developmentally critical protein, glial cell line-derived neurotrophic factor (GDNF), could improve graft outcomes. We tracked the response of DAns implanted into either a GDNF-rich environment or after a delay in exposure. Early GDNF promoted survival and plasticity of non-DAns, leading to enhanced motor recovery in PD rats. Delayed exposure to GDNF promoted functional recovery through increases in DAn specification, DAn plasticity, and DA metabolism. Transcriptional profiling revealed a role for mitogen-activated protein kinase (MAPK)-signaling downstream of GDNF. Collectively, these results demonstrate the potential of neurotrophic gene therapy strategies to improve hPSC graft outcomes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-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 using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.

The levels and conformers of alpha-synuclein are critical in the pathogenesis of Parkinson's Disease and related synucleinopathies. Homeostatic mechanisms in protein degradation and secretion have been identified as regulators of alpha-synuclein at different stages of its intracellular trafficking and transcellular propagation. Here we review pathways involved in the removal of various forms of alpha-synuclein from both the intracellular and extracellular environment. Proteasomes and lysosomes are likely to play complementary roles in the removal of intracellular alpha-synuclein species, in a manner that depends on alpha-synuclein post-translational modifications. Extracellular alpha-synuclein is cleared by extracellular proteolytic enzymes, or taken up by neighboring cells, especially microglia and astrocytes, and degraded within lysosomes. Exosomes, on the other hand, represent a vehicle for egress of excess burden of the intracellular protein, potentially contributing to the transfer of alpha-synuclein between cells. Dysfunction in any one of these clearance mechanisms, or a combination thereof, may be involved in the initiation or progression of Parkinson's disease, whereas targeting these pathways may offer an opportunity for therapeutic intervention. This article is part of the Special Issue "Synuclein"., (© 2019 International Society for Neurochemistry.)

One of the most robust neurochemical abnormalities reported in patients living with schizophrenia is an increase in dopamine (DA) synthesis and release in the dorsal striatum (DS). Importantly, it appears that this increase progresses as a patient transitions from a prodromal stage to the clinical diagnosis of schizophrenia. Here we have recreated this pathophysiology in an animal model by increasing the capacity for DA synthesis preferentially within the DS. To achieve this we administer a genetic construct containing the rate-limiting enzymes in DA synthesis-tyrosine hydroxylase (TH), and GTP cyclohydrolase 1 (GCH1) (packaged within an adeno-associated virus)-into the substantia nigra pars compacta (SNpc) of adolescent animals. We refer to this model as "Enhanced Dopamine in Prodromal Schizophrenia" (EDiPS). We first confirmed that the TH enzyme is preferentially increased in the DS. As adults, EDiPS animals release significantly more DA in the DS following a low dose of amphetamine (AMPH), have increased AMPH-induced hyperlocomotion and show deficits in pre-pulse inhibition (PPI). The glutamatergic response to AMPH is also altered, again in the DS. EDiPS represents an ideal experimental platform to (a) understand how a preferential increase in DA synthesis capacity in the DS relates to "positive" symptoms in schizophrenia; (b) understand how manipulation of DS DA may influence other neurotransmitter systems shown to be altered in patients with schizophrenia; (c) allow researchers to follow an "at risk"-like disease course from adolescence to adulthood; and (d) ultimately allow trials of putative prophylactic agents to prevent disease onset in vulnerable populations.

Parkinson's disease is characterized by a progressive loss of substantia nigra (SN) dopaminergic neurons and the formation of Lewy bodies containing accumulated alpha-synuclein (α-syn). The pathology of Parkinson's disease is associated with neuroinflammatory microglial activation, which may contribute to the ongoing neurodegeneration. This study investigates the in vivo microglial and dopaminergic response to overexpression of α-syn. We used positron emission tomography (PET) and the 18 kDa translocator protein radioligand, [ 11 C](R)PK11195, to image brain microglial activation and (+)-α-[ 11 C]dihydrotetrabenazine ([ 11 C]DTBZ), to measure vesicular monoamine transporter 2 (VMAT2) availability in Göttingen minipigs following injection with recombinant adeno-associated virus (rAAV) vectors expressing either mutant A53T α-syn or green fluorescent protein (GFP) into the SN (4 rAAV-α-syn, 4 rAAV-GFP, 5 non-injected control minipigs). We performed motor symptom assessment and immunohistochemical examination of tyrosine hydroxylase (TH) and transgene expression. Expression of GFP and α-syn was observed at the SN injection site and in the striatum. We observed no motor symptoms or changes in striatal [ 11 C]DTBZ binding potential in vivo or striatal or SN TH staining in vitro between the groups. The mean [ 11 C](R)PK11195 total volume of distribution was significantly higher in the basal ganglia and cortical areas of the α-syn group than the control animals. We conclude that mutant α-syn expression in the SN resulted in microglial activation in multiple sub- and cortical regions, while it did not affect TH stains or VMAT2 availability. Our data suggest that microglial activation constitutes an early response to accumulation of α-syn in the absence of dopamine neuron degeneration., (© 2018 Wiley Periodicals, Inc.)

Impairment of the ubiquitin proteasome system has been implicated in Parkinson's disease. We used positron emission tomography to investigate longitudinal effects of chronic intracerebroventricular exposure to the proteasome inhibitor lactacystin on monoaminergic projections and neuroinflammation. Göttingen minipigs were implanted in the cisterna magna with a catheter connected to a subcutaneous injection port. Minipigs were imaged at baseline and after cumulative doses of 200 and 400 μg lactacystin, respectively. Main radioligands included [ 11 C]-DTBZ (vesicular monoamine transporter type 2) and [ 11 C]-yohimbine (α2-adrenoceptor). [ 11 C]-DASB (serotonin transporter) and [ 11 C]-PK11195 (activated microglia) became available later in the study and we present their results in a smaller subset of animals for information purposes only. Striatal [ 11 C]-DTBZ binding potentials decreased significantly by 16% after 200 μg compared to baseline, but the decrease was not sustained after 400 μg (n = 6). [ 11 C]-yohimbine volume of distribution increased by 18-25% in the pons, grey matter and the thalamus after 200 μg, which persisted at 400 μg (n = 6). In the later subset of minipigs, we observed decreased [ 11 C]-DASB (n = 5) and increased [ 11 C]-PK11195 (n = 3) uptake after 200 μg. These changes may mimic monoaminergic changes and compensatory responses in early Parkinson's disease.

The neurodegenerative Huntington's disease (HD) is caused by a polyglutamine (polyQ) amplification in the huntingtin protein (HTT). Currently there is no effective therapy available for HD; however, several efforts are directed to develop and optimize HTT-lowering methods to improve HD phenotypes. To validate these approaches, there is an immediate need for reliable, sensitive, and easily accessible methods to quantify HTT expression. Using the AlphaLISA platform, we developed two novel sensitive and robust assays for quantification of HTT in biological samples using commercially available antibodies. The first, a polyQ-independent assay, measures the total pool of HTT, while the second, a polyQ-dependent assay, preferentially detects the mutant form of HTT. Using purified HTT protein standards and brain homogenates from an HD mouse model, we determine a lower limit of quantification of 1 and 3 pm and optimal reproducibility with CV values lower than 7% for intra- and 20% for interassay. In addition, we used the assays to quantify HTT in neural stem cells generated from patient-derived induced pluripotent stem cells in vitro and in human brain tissue lysates. Finally, we could detect changes in HTT levels in a mouse model where mutant HTT was conditionally deleted in neural tissue, verifying the potential to monitor the outcome of HTT-lowering strategies. This analytical platform is ideal for high-throughput screens and thus has an added value for the HD community as a tool to optimize novel therapeutic approaches aimed at modulating HTT protein levels.

Parvalbumin- (PV-) containing basket cells constitute perisomatic GABAergic inhibitory interneurons innervating principal cells at perisomatic area, a strategic location that allows them to efficiently control the output and synchronize oscillatory activity at gamma frequency (30-90 Hz) oscillations. This oscillatory activity can convert into higher frequency epileptiform activity, and therefore could play an important role in the generation of seizures. However, the role of endogenous modulators of seizure activity, such as Neuropeptide Y (NPY), has not been fully explored in at PV input and output synapses. Here, using selective optogenetic activation of PV cells in the hippocampus, we show that seizures, induced by rapid kindling (RK) stimulations, enhance gamma-aminobutyric acid (GABA) release from PV cells onto dentate gyrus (DG) granule cells (GC). However, PV-GC synapses did not differ between controls and kindled animals in terms of GABA release probability, short-term plasticity and sensitivity to NPY. Kinetics of gamma-aminobutyric acid A (GABA-A) mediated currents in postsynaptic GC were also unaffected. When challenged by repetitive high-frequency optogenetic stimulations, PV synapses in kindled animals responded with enhanced GABA release onto GC. These results unveil a mechanism that might possibly contribute to the generation of abnormal synchrony and maintenance of epileptic seizures.