Biology subjects

Jensen, P. H.

Publications and source records attributed to Jensen, P. H..

16 recordsLinked to original sources

bioRxiv2025

Lack of functional STING modulates immunity but does not protect dopaminergic neurons in the alpha-synuclein preformed fibrils Parkinson's Disease mouse model

Microglia response is proposed to be relevant in the neurogenerative process associated with alpha-synuclein (-syn) pathology in Parkinsons disease (PD). STING is a protein related to the immune sensing of DNA and autophagy, and it has been proposed to be involved in PD neurodegeneration. To investigate this, we injected 10 {micro}g of murine pre-formed fibrils (PFFs) of -syn (or monomeric and PBS as controls) into the striatum of wild-type (WT) and STINGgt/gt mice, which lack functional STING. We examined motor behavior and brain pathology at 1- and 6-months post-injection. STINGgt/gt mice showed more motor changes associated with PFF injection than WT mice. STINGgt/gt mice had a differential immune response to PFF with early and sustained increased microglia numbers and higher macrophagic CD68 response, but milder changes in the expression of immune-relevant markers such as TLR2, TLR4, IL1b, and TREM2. However, the lack of STING did not induce changes in the extent of -syn pathology nor the p62 accumulation seen in the model. Altogether, this resulted in a faster but similar degree of nigrostriatal dopaminergic degeneration after 6 months. Therefore, the data do not support a necessary role for STING in the -syn induced nigral neuronal loss in the PFF-PD mouse model used here. However, the results suggest a functional relevance for STING in the brain response to the excess and aggregation of amylogenic proteins such as -syn that can contribute to symptomatic changes.

neuroscience↗

bioRxiv2025

Human- and Rodent-derived Extracellular Vesicles Mediate the Spread of Pathology in MSA-like Models

Multiple system atrophy (MSA) is characterized by the presence of protein-rich inclusions mainly within oligodendrocytes, comprised primarily by the neuronal protein Synuclein and the oligodendroglial-specific phosphoprotein TPPP/p25. Mature oligodendrocytes do not normally express detectable Synuclein levels, suggesting that its oligodendroglial accumulation may arise from intercellular transfer, potentially via extracellular vesicles (EVs); however the precise role of oligodendroglial-derived EVs in MSA progression remains relatively understudied. Herein, we characterized the cargo/features and pathogenic potential of EVs released by oligodendrocytes treated with human Synuclein fibrils amplified from MSA or Parkinsons disease patient brains (or human recombinant Synuclein fibrils) and EVs isolated from murine and human MSA (or respective control) brains. Our findings reveal that both oligodendroglial cell- and brain-derived EVs harbor pathological Synuclein and TPPP/p25 conformations, similar to those accumulating in human MSA brains. These EVs are readily taken up by both neurons and oligodendrocytes, driving Synuclein propagation in vitro. Importantly, inoculation of these MSA-like EVs in animal models induce robust pSer129-Synuclein accumulation along the nigrostriatal axis, colocalizing with markers of mature oligodendrocytes and dopaminergic neurons. These findings underscore the pivotal role of oligodendroglial-derived EVs in pathology progression and neuronal-oligodendroglial communication, positioning them as promising targets for therapeutic strategies aimed at combating alpha-Synucleinopathies.

neuroscience↗

bioRxiv2024

Abundant non-inclusion α-synuclein pathology in Lewy body-negative LRRK2-mutant cases

Lewy body diseases are common neurodegenerative diseases, including Parkinsons disease (PD) and dementia with Lewy bodies, which lead to both motor and non-motor symptoms. They are neuropathologically characterized by loss of neuromelanized neurons in the substantia nigra pars compacta and -synuclein-immunopositive inclusions (Lewy bodies) in several types of neurons in the brain. A fraction of monogenic PD cases, however, represent a conundrum, as they can present with clinical Lewy body disease but do not have Lewy bodies upon neuropathological examination. For LRRK2, the presence or absence of Lewy bodies is not related to any specific mutation in the gene and different clinical presentation and neuropathology can be present even in the same family. Here, we present the first evidence of widespread -synuclein accumulation detected with proximity ligation assay (PLA) using the MJFR14-6-4-2 antibody in six Lewy body-negative LRRK2 cases and compare the levels with five patients with neuropathologically-verified Lewy body disease and six healthy controls. We show that non-inclusion aggregated -synuclein in the form of particulate PLA signal is dominant in the LRRK2 cases, while both Lewy-like and particulate PLA signal is found in late-stage Lewy body disease. Furthermore, LRRK2 cases displayed prominent particulate PLA signal in pontocerebellar tracts and inferior olivary nuclei in the brainstem, which was not seen in idiopathic Lewy body disease cases. These results suggest that Lewy-body negative LRRK2-related PD is not associated with a lack of -synuclein aggregation in neurons but rather a deficiency in the formation of inclusions.

neuroscience↗

bioRxiv2024

Molecular signatures of altered energy metabolism and circadian rhythm perturbations in a model of extra-nigral Synucleinopathy.

A pathological role of alpha-Synuclein (aSyn) aggregation in the central nervous system (CNS) is a recognized feature in Parkinson disease (PD) and related neurodegenerative conditions termed synucleinopathies. In order to characterize the cellular response in CNS to incipient and advanced aSyn pathology, we applied spatial transcriptomics on brain sections derived from a transgenic mouse model (M83+/+ line, Prnp-SNCA*A53T) in which aSyn aggregation was induced in a prion-like fashion through hindlimb intramuscular delivery of pre-formed fibrillar (PFF) murine aSyn. Our spatially-resolved transcriptomics (ST) data point to unique perturbations in brain energy metabolism during the progression of aSyn pathology, such that the early stage of aSyn aggregate pathology activates molecular pathways controlling metabolic flux through glycolysis, oxidative phosphorylation and fatty acid metabolism. In contrast, the ST data indicate a profound decline in mitochondrial metabolism in the brains of symptomatic animals with advanced aSyn pathology. The latter stage was also associated with drastic reduction in mRNA translation machinery, along with aberrant expression of molecular drivers involved in RNA splicing and inflammatory response. Intriguingly, our ST data also point to perturbed regulation of circadian rhythm, was corroborated by increased immunodetection of CREB-binding protein (a modulator of core clock machinery) in the brains of symptomatic animals, and transcriptional upregulation of CREBBP in 4 independent PD microarray datasets. Collectively, we anticipate that our findings offer novel opportunities in knowledge translation for mechanism-based drug discovery and biomarkers in neurodegenerative synucleinopathies.

neuroscience↗

bioRxiv2024

Bradykinesia and postural instability in a model of prodromal Synucleinopathy with alpha-Synuclein aggregation in the gigantocellular nuclei.

-Synuclein (aSyn) accumulation within the extra-nigral neuronal populations in brainstem, including the gigantocellular nuclei (GRN/Gi) of reticular formation, is a recognized feature during the prodromal phase of Parkinson disease (PD). Accordingly, there is a burgeoning interest in animal model development for understanding the pathological significance of extra-nigral synucleinopathy, in relation to motor and/or non-motor symptomatology in PD. Here, we report an experimental paradigm for the induction of aSyn aggregation in brainstem, with stereotaxic delivery of pre-formed fibrillar (PFF) aSyn in the pontine GRN of transgenic mice expressing the mutant human Ala53Thr aSyn (M83 line). Our data show that PFF aSyn-induced aggregate pathology in GRN leads to progressive decline in spontaneous locomotion and an early phenotype of postural instability. This early phase of bradykinesia was followed by a moribund stage, characterized by worsening motor performance and impaired survival with substantial aSyn aggregation in several brain regions beyond the GRN. Collectively, our observations suggest an experimental framework for studying the pathological significance of aSyn aggregation in GRN in relation to features of movement disability in PD. With further refinements, we anticipate that this model holds promise as a test-bed for translational research in PD and related disorders.

neuroscience↗

bioRxiv2024

MJF-14 proximity ligation assay detects early non-inclusion alpha-synuclein pathology with enhanced specificity and sensitivity

Lewy pathology, consisting of Lewy bodies and Lewy neurites, is the pathological hallmark of synucle-inopathies such as Parkinsons disease and dementia with Lewy bodies, but it is generally thought to represent late-stage pathological changes. In contrast, -synuclein oligomers are regarded as early-stage pathology, likely involved in disease progression and cellular toxicity. Oligomers, however, are not de-tected by standard immunohistochemistry but require specific detection techniques such as the proxim-ity ligation assay (PLA). Here, we describe the MJF-14 PLA, a new PLA towards aggregated -synuclein with unprecedented specificity, attained by the utilization of aggregate conformation-specific -synu-clein antibody MJFR-14-6-4-2 (hereafter MJF-14). Signal in the assay directly correlates with -synuclein aggregation in SH-SY5Y cells, as treatment with aggregation inhibitor ASI1D significantly lowers PLA sig-nal. In human cortical neurons, MJF-14 PLA detects pre-formed fibril-induced aggregation, especially prominent when using stealth PFFs invisible to the MJF-14 antibody. Co-labelling of MJF-14 PLA and pS129--synuclein immunofluorescence in post-mortem dementia with Lewy bodies cases showed that while the MJF-14 PLA reveals extensive non-inclusion pathology, it is not sensitive towards Lewy bodies. In Parkinsons disease brain, direct comparison of PLA and IHC with the MJF-14 antibody, combined with machine learning-based quantification, showed striking -synuclein pathology preceding the formation of conventional Lewy pathology. The majority of the PLA-revealed non-inclusion pathology was found in the neuropil, including some clearly located in the presynaptic terminals. With this work, we introduce an improved -synuclein aggregate PLA to uncover abundant non-inclusion pathology, which deserves future validation with multiple brain bank resources and in different synucleinopathies.

neuroscience↗

bioRxiv2024

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 [≤]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↗

bioRxiv2023

Structural basis of epitope recognition by anti-alpha synuclein antibodies MJFR14-6-4-2

Intraneuronal -synuclein inclusions in the brain are hallmarks of so-called Lewy body diseases - Parkinsons disease and Dementia with Lewy bodies. Lewy bodies are cytoplasmic inclusions, containing mainly aggregated -synuclein together with some other proteins including ubiquitin, neurofilament protein, and alpha B crystallin. In its monomeric form, -synuclein is predominantly localized in nerve terminals, regulating neuronal transmission and synaptic vesicle trafficking. Monomeric -synuclein lacks a well-defined three-dimensional structure and is considered an intrinsically disordered protein. However, in diseased cells -synuclein aggregates into oligomeric and fibrillar amyloid species, which can be detected using aggregate-specific antibodies. Here we investigate the aggregate specificity of rabbit monoclonal MJFR14-6-4-2 antibodies, preferentially recognizing aggregated -synuclein species. We conclude that partial masking of epitope in unstructured monomer in combination with a high local concentration of epitopes instead of distinct epitope conformation is the main reason for apparent selectivity towards various aggregates, including oligomers, fibrils, and artificial virus-like particle constructs bearing multiple copies of the MJFR14-6-4-2 epitope. Based on the structural insight, we were able to express mutant -synuclein that when fibrillated are unable to bind MJFR14-6-4-2. Using these "stealth" fibrils as a tool for seeding cellular -synuclein aggregation, provides superior signal/noise ratio for detection of cellular -synuclein aggregates by MJFR14-6-4-2 immunocytochemistry. Our data provide a molecular level understanding of specific recognition of toxic amyloid oligomers, which is critical for the development of inhibitors against synucleinopathies.

neuroscience↗

bioRxiv2022

Sex-dimorphic neuroprotective effect of CD163 in an alpha-synuclein mouse model Of Parkinson's disease

Alpha-synuclein (-syn) aggregation and immune activation represent hallmark pathological events in Parkinsons disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in the PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine -syn pre-formed fibrils (PFF) in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to -syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and -syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a novel, sex-dimorphic neuroprotective role for CD163 during -syn-induced neurodegeneration.

neuroscience↗

bioRxiv2022

Monomeric α-Synuclein activates the Plasma Membrane Calcium Pump

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well-known for pathological aggregation in neurodegeneration. The physiological function of aSN however is disputed. Pull-down experiments have pointed to plasma membrane Ca2+-ATPase (PMCA) as a potential interaction partner. From proximity ligation assays we find that aSN and PMCA colocalize at neuronal synapses, and that calcium expulsion is activated by aSN and PMCA. From PMCA activity studies we show that soluble, monomeric aSN activates PMCA at par with CaM, yet independent of the autoinhibitory domain of PMCA, but highly dependent on acidic phospholipids and membrane-anchoring of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop region connecting the cytosolic A domain and transmembrane segment 3. Our studies point towards a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

biochemistry↗

bioRxiv2021

Polarized α-synuclein trafficking and transcytosis across Brain Endothelial Cells via Rab7-decorated carriers

Parkinsons disease is mainly caused by aggregation of -synuclein (-syn) in the brain. Exchange of -syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of -syn across the blood brain barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of -syn monomers and oligomers across an in vitro BBB model system. Both -syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric -syn in brain endothelial cells, we investigated co-localization of -syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric -syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric -syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric -syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric -syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric -syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinsons disease and related synucleinopathies. Significance StatementIn the submitted manuscript, we describe the use of a state-of-the-art porcine blood-brain barrier model based on primary cells to get information about this important issue. We identify several hitherto undescribed cellular pathways to mediate polarized transport of alpha-synuclein. One of these paths we find regulated by Rab7 and can be inhibited by targeting several intracellular proteins such as VPS35, Caveolin1 and Rab7. New knowledge describing brain endothelial intracellular transport systems are highly warranted for identifying new target to alleviate Parkinsons disease. We believe that our findings could be the seed to establish new therapeutic strategies against Parkinsons disease and related synucleinopathies.

cell biology↗

bioRxiv2021

Polo-like kinase 2 inhibition reduces serine-129 phosphorylation of physiological nuclear alpha-synuclein but not of the aggregated alpha-synuclein

Accumulation of aggregated alpha-synuclein (-syn) is believed to play a pivotal role in the pathophysiology of Parkinsons disease (PD) and other synucleinopathies. -Syn is a key constituent protein of Lewy pathology, and -syn phosphorylated at serine-129 (pS129) constitutes more than 90% of -syn in Lewy bodies and hence, it is used extensively as a pathological marker for the aggregated form of -syn. However, the exact role of pS129 remains controversial as well as the kinase(s) responsible for the phosphorylation. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using ex-vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on -syn aggregation, pS129 or inter-neuronal spreading of the aggregated -syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble nuclear pS129 level confined in the nuclei. The same finding was replicated in an in-vivo mouse models of templated -syn aggregation and human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129 phosphorylation of soluble non-pathology related fraction of -syn. We also demonstrated that reduction of nuclear pS129 but not the aggregates specific pS129 following PLK2 inhibition for a short time before sample collection improves the signal to noise ratio when quantifying pS129 aggregate pathology.

neuroscience↗

bioRxiv2021

α-Synuclein pathology in Parkinson disease activates homeostatic NRF2 anti-oxidant response

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleionpathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2) is recognized as the master regulator of cellular anti-oxidant response, both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, Prnp-SNCA*Ala53Thr), which manifest widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

neuroscience↗

bioRxiv2019

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↗

bioRxiv2019

Stabilization of α-synuclein oligomers using formaldehyde

The group of neurodegenerative diseases, Parkinsons disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type -synuclein (-syn) aggregates within degenerating brain cells. -syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating -syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized -syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific -syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked -syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked -syn oligomers could serve as important calibrators to facilitate comparative and standardized -syn biomarker studies going forward.

neuroscience↗

bioRxiv2018

Long-term exposure to PFE-360 in the AAV-α-synuclein rat model: findings and implications

Significance statementTreatment of Parkinsons disease is reliant on symptomatic treatments, without any option to slow or halt disease progression. Mutations in LRRK2 and -synuclein are known risk factors for Parkinsons disease. Presence of -synuclein aggregates at autopsies in both idiopathic and most G2019S cases is suggestive of a common disease pathogenesis. LRRK2 and -synuclein interaction is hypothesized to play a pivotal role in the pathological mechanisms and inhibitors of LRRK2 are investigated as novel disease modulatory treatments in the clinic. However, preclinical in vivo evidence of a beneficial effect of LRRK2 inhibition is mixed and limited. This study increases our understanding of LRRK2 as a mediator of neuronal dysfunction and the potential of LRRK2 as a promising target in PD.

neuroscience↗