CSF protein dynamics in murine models of α-synucleinopathy and cerebral β-amyloidosis

Parkinson’s (PD) and Alzheimer’s disease (AD) are the two most common neurodegen- erative diseases and of growing importance for the rapidly aging population of indus- trialized countries. A common feature of both diseases is the progressive accumulation of proteins in insoluble aggregates, which are considered to play a fundamental role in the pathogeneses ultimately resulting in marked neuronal loss. The histopathological hallmark features of PD are called Lewy bodies (LBs) and Lewy neurites (LNs), both consisting predominantly of aggregated a-synuclein (aSyn), while neuropathological di- agnosis of AD relies on the presence of neurofibrillary tangles and senile plaques com- prised of hyperphosphorylated tau-protein or amyloid-b (Ab), respectively. However, the implicated pathological mechanisms and pathways contributing to these diseases are still not fully understood and numerous studies emphasize the need for early diagnosis before major neuron-loss occurs. Cerebrospinal fluid (CSF) is in close contact with the central nervous system and therefore a valuable source of biochemical markers reflecting pathological changes in the brain and spinal cord. For AD, three core CSF biomarkers (Ab, total-tau & phospho-tau) have been identified and extensively validated over the last years, while established biochemical markers are virtually absent for the diagnosis of PD. The aim of the thesis was to investigate proteome alteration in the presence of different types of protein aggregates and ultimately to identify novel biochemical markers of disease. Therefore, transgenic mouse models of a-synucleinopathy or b-amyloidosis, the hallmark neuropathological aspects of PD or AD, were used. These mice express mutated human genes initially identified in patients suffering from familial forms of PD or AD. The first set of experiments focused on the identification of proteins altered in the CSF of these models. This was done in an unbiased mass spectrometry-based shotgun approach, which led to the quantification of 636 and 665 CSF proteins in aged A30P- aSyn and APPPS1 cohorts, respectively. Both datasets contained transgene-related CSF protein changes that have already been associated with PD or AD, such as amy- loid precursor protein (APP)-derived peptides, TREM2, ApoE or neurofilament light (NfL), but also yielded novel insights in protein alterations, such as LAG-3, CART and lysosomal proteins. Next, both datasets generated from A30P-aSyn and APPPS1 CSF were compared and revealed a marked overlap of proteins deregulated in both models. The second part of the thesis focused on NfL, which plays a key role in axonal sta- bilization and gained attention as biomarker of axonal injury in multiple neurological disorders. A validated immunoassay was used for the quantification of NfL in CSF and plasma of A30P-aSyn, APPPS1 and a third mouse line expressing an A53T mutation in aSyn. Markedly elevated levels were found in CSF and plasma of the mice at the same age as the respective brain lesions became apparent. This demonstrates that CSF and blood NfL increases are not specific for aggregated aSyn or b-amyloid and emphasizes its potential as marker of axonal damage upon neurodegeneration. In conclusion, hundreds of proteins were quantified in the CSF of mouse models for a-synucleinopathy and b-amyloidosis. The datasets at hand provide novel and unbiased insights in pathological processes on molecular level and reveal common and distinct features of the respective pathology. The high proportion of hits related to PD, AD and other neurodegenerative diseases, as evaluated in human-based studies, substan- tiates the confidence in the high quality of the datasets and the translational value of the mouse models. Taken together, these findings provide a rich resource for the identification of novel biomarkers and their value concerning differential diagnosis

LAG3 is not expressed in human and murine neurons and does not modulate α-synucleinopathies.

While the initial pathology of Parkinson's disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid

Quantitative Phosphoproteomics of Murine <i>Fmr1</i>-KO Cell Lines Provides New Insights into FMRP-Dependent Signal Transduction Mechanisms

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the <i>FMR1</i> gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine <i>FMR1</i>^– and <i>FMR1</i>⁺ fibroblastic cell lines derived from <i>FMR1</i>-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS

LAG3 is not expressed in human and murine neurons and does not modulate α‐synucleinopathies

While the initial pathology of Parkinson’s disease and other α-synucleinopathies is often confined to circumscribed brain regions, it can spread and progressively affect adjacent and distant brain locales. This process may be controlled by cellular receptors of α-synuclein fibrils, one of which was proposed to be the LAG3 immune checkpoint molecule. Here, we analysed the expression pattern of LAG3 in human and mouse brains. Using a variety of methods and model systems, we found no evidence for LAG3 expression by neurons. While we confirmed that LAG3 interacts with α-synuclein fibrils, the specificity of this interaction appears limited. Moreover, overexpression of LAG3 in cultured human neural cells did not cause any worsening of α-synuclein pathology ex vivo. The overall survival of A53T α-synuclein transgenic mice was unaffected by LAG3 depletion, and the seeded induction of α-synuclein lesions in hippocampal slice cultures was unaffected by LAG3 knockout. These data suggest that the proposed role of LAG3 in the spreading of α-synucleinopathies is not universally valid

Mapping the upper mantle: three-dimensional modeling of Earth structure by inversion of seismic waveforms.

A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases

Signatures of glial activity can be detected in the CSF proteome

Single-cell transcriptomics has revealed specific glial activation states associated with the pathogenesis of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. While these findings may eventually lead to new therapeutic opportunities, little is known about how these glial responses are reflected by biomarker changes in bodily fluids. Such knowledge, however, appears crucial for patient stratification, as well as monitoring disease progression and treatment responses in clinical trials. Here, we took advantage of well-described mouse models of beta-amyloidosis and alpha-synucleinopathy to explore cerebrospinal fluid (CSF) proteome changes related to their respective proteopathic lesions. Nontargeted liquid chromatography-mass spectrometry revealed that the majority of proteins that undergo age-related changes in CSF of either mouse model were linked to microglia and astrocytes. Specifically, we identified a panel of more than 20 glial-derived proteins that were increased in CSF of aged beta-amyloid precursor protein- and alpha-synuclein-transgenic mice and largely overlap with previously described disease-associated glial genes identified by single-cell transcriptomics. Our results also show that enhanced shedding is responsible for the increase of several of the identified glial CSF proteins as exemplified for TREM2. Notably, the vast majority of these proteins can also be quantified in human CSF and reveal changes in Alzheimer's disease cohorts. The finding that cellular transcriptome changes translate into corresponding changes of CSF proteins is of clinical relevance, supporting efforts to identify fluid biomarkers that reflect the various functional states of glial responses in cerebral proteopathies, such as Alzheimer's and Parkinson's disease