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

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.

Image post-processing for SILMAS: structured illumination light sheet microscopy with axial sweeping.

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.

State of the Art in Sub-Phenotyping Midbrain Dopamine Neurons.

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.

Basal activity of PINK1 and PRKN in cell models and rodent brain.

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 the brain remain undetermined.

Generation of the iPSC line FINi002-A from a male Parkinson's disease patient carrying compound heterozygous mutations in the PRKN gene.

The most common cause of autosomal recessive familial Parkinson's disease (PD) are mutations in the PRKN/PARK2 gene encoding an E3 ubiquitin protein-ligase PARKIN. We report the generation of an iPSC cell line from the fibroblasts of a male PD patient carrying a common missense variant in exon 7 (p.Arg275Trp), and a 133 kb deletion encompassing exon 8, using transiently-present Sendai virus.

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

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.

Generation of human-induced pluripotent-stem-cell-derived cortical neurons for high-throughput imaging of neurite morphology and neuron maturation.

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.

Effects of mutant huntingtin in oxytocin neurons on non-motor features of Huntington's disease.

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.

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

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.

Aging, Parkinson's Disease, and Models: What Are the Challenges?

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.

High contrast, isotropic, and uniform 3D-imaging of centimeter-scale scattering samples using structured illumination light-sheet microscopy with axial sweeping.

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.

Combining CRISPR-Cas9 and brain imaging to study the link from genes to molecules to networks.

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.

A combined cell and gene therapy approach for homotopic reconstruction of midbrain dopamine pathways using human pluripotent stem cells.

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.

DNAJB6 suppresses alpha-synuclein induced pathology in an animal model of Parkinson's disease.

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.

Comparison of Locus Coeruleus Pathology with Nigral and Forebrain Pathology in Parkinson's Disease.

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.

Positive symptom phenotypes appear progressively in "EDiPS", a new animal model of the schizophrenia prodrome.

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.

Effects of mutant huntingtin inactivation on Huntington disease-related behaviours in the BACHD mouse model.

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.

Viral-based rodent and nonhuman primate models of multiple system atrophy: Fidelity to the human disease.

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.

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

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.

Viral Delivery of GDNF Promotes Functional Integration of Human Stem Cell Grafts in Parkinson's Disease.

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.

Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates.

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.

How is alpha-synuclein cleared from the cell?

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.

Enhanced Dopamine in Prodromal Schizophrenia (EDiPS): a new animal model of relevance to schizophrenia.

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.

Longitudinal monoaminergic PET imaging of chronic proteasome inhibition in minipigs.

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.