Distribution of pathological α -synuclein in the CNS of diseased mice

The influence of the different inoculation routes on α-synuclein aggregation and spreading in the CNS was investigated by analysis of different brain and spinal cord tissue sections with the pSyn#64 antibody recognizing phosphorylation at S129 of α-synuclein and an additional counterstaining with hematoxylin as a nuclear marker (Figure 3.4). Immunostaining visualized a strong accumulation of pathological α-synuclein throughout the whole brain and displayed a rostral to caudal pattern of pathology intensity in TgM83^+/− mice for every inoculation route. Pathology was detected regardless of the route of inoculation in the frontal lobe, such as the motor cortex and striatum, in the parietal-temporal lobe, including the amygdala and the hypothalamus, and in the brain stem of challenged animals. Phosphorylated α-synuclein was present in neuronal cell bodies and neurites within these brain areas and the spinal cord of all mice challenged with α-synuclein fibrils. Interestingly, the hippocampus only showed α-synuclein pathology after intracerebral or oral challenge with α-synuclein fibrils but was not affected after intraperitoneal or intravenous

Results Peripheral challenge study

injection. None of the BSA-challenged control mice showed deposits of phosphorylated α-synuclein in any of these brain areas or the spinal cord.

Results Peripheral challenge study

Figure 3.4: Immunohistochemical analysis shows neuropathology in the CNS of TgM83^+/−

mice after challenge with α-synuclein fibrils for all four routes. In TgM83^+/− mice challenged intracerebrally, intraperitoneally, intravenously, or orally with α-synuclein (α-syn) fibrils, phosphorylated α-synuclein was present in neuronal cell bodies and neurites, as detected by staining with the pSyn#64 antibody recognizing phosphorylation at S129 of α-synuclein.

Pathological α-synuclein was present throughout the brain, including the motor cortex, striatum, hippocampus, hypothalamus, amygdala, brain stem, as well as in the spinal cord.

In contrast, none of the BSA-challenged mice showed any phosphorylated α-synuclein in the brain or the spinal cord. Scale bar = 50 μm. Published in Lohmann et al., 2019.

Anatomical distribution of pathology in the form of phosphorylated α-synuclein deposits across the four inoculation groups was quantified. The findings were scaled from 0 to 5 reflective of no to severe pathology, and depicted as a heat map showing the extent of pathology in different brain regions (Figure 3.5). The most frontal sections at bregma 0.26 mm with mostly cortical regions showed none or just sparse pathology. Most pathology was detected in the primary and secondary motor cortex, where more affected neurites and somata were present in all four groups. Pathological α-synuclein was detected in the striatum and the lateral septal nucleus for all inoculation routes, with minor pathology presented after intraperitoneal challenge. At bregma –1.70 mm, affected neurites were spread throughout most of the cortical regions, showing less pathology after intravenous injection. A nearly identical amount of α-synuclein pathology was observed in the interbrain regions for all transmission routes. Here, the brains showed a sparse to mild pathology in the amygdala, a mild pathology throughout the thalamus, and a moderate pathology throughout the hypothalamus. Dense pathology was observed in the peduncular part of the lateral hypothalamus after oral and intravenous challenge. The hippocampal regions were not affected after intravenous or intraperitoneal challenge. However, sparse pathology was detected in all hippocampal regions after oral challenge. After intracerebral challenge mild pathology was detected in the CA1 region, moderate pathology in the CA3 region, and a severe pathology in the dentate gyrus. For sections from posterior areas, at bregma –3.28 mm no pathology was detected in any of the cortical regions for all but intracerebrally challenged mice, where sparse pathology was found in most cortical regions. Distribution of α-synuclein within the hippocampal regions showed sparse pathology for the oral route, and sparse to mild pathology in the CA1 and CA3 region, and moderate pathology in the dentate gyrus for the intracerebral route. An increased amount of pathology was observed in the midbrain region of all four groups, with sparse-to-mild pathology in the medial geniculate nuclei and a mild-to-moderate pathology in the parabrachial pigmented nucleus of the ventral tegmental area. The substantia nigra displayed a mild pathology after oral, intracerebral, and intraperitoneal challenge with α-synuclein fibrils and a moderate pathology was detected after intravenous injection. The superior colliculus indicated the following distribution pattern: mild-to-dense for intraperitoneal and intracerebral challenge, moderate-to-dense for intravenous challenge, and moderate-to-severe pathology for orally challenged mice. In the inner midbrain regions consistent moderate-to-dense pathology was observed in the lateral periaqueductal grey, the anterior pretectal

Results Peripheral challenge study

nucleus, the parvicellular part of the red nucleus, and the mesencephalic reticular formation consistent in all four groups. The only exception was a severe pathology in the mesencephalic reticular formation of orally challenged mice. At bregma –6.24 mm sparse-to-mild pathology was observed in the cerebellar nuclei, except for the cerebellum. Sparse pathology was also detected in the outer regions of the hindbrain, such as the inferior cerebellar peduncle and the spinal trigeminal tract. In contrast, moderate-to-dense pathology was present in the facial and the raphe magnus nuclei in all groups. Dense pathology was observed in the reticular nucleus after oral, intracerebral, and intraperitoneal challenge, and severe pathology after intravenous challenge.

Figure 3.5: Heat map showing the distribution of phosphorylated α-synuclein in TgM83^+/−

mice after challenge with α-synuclein fibrils for all four routes. Distribution of phosphorylated α-synuclein in the brains of inoculated TgM83^+/− mice with human α-synuclein fibrils was quantified and summarized on a scale from 0 to 5 reflective of no to severe pathology as a heat map for four coronal brain sections. n = 3/group. Published in Lohmann et al., 2019.

Results Peripheral challenge study

Generally, oral, intracerebral, intraperitoneal, and intravenous challenge resulted in a consistent distribution of phosphorylated α-synuclein deposits but with overall less pathology after intraperitoneal challenge. One exception was the hippocampus, especially the dentate gyrus, showing pathology only after intracerebral and oral challenge but not after intravenous or intraperitoneal challenge with α-synuclein fibrils.

3.1.4 Neuropathology displays oligomeric and fibrillar α-synuclein