Long‐lasting pathological consequences of overexpression‐induced α‐synuclein spreading in the rat brain

Summary Increased expression of α‐synuclein can initiate its long‐distance brain transfer, representing a potential mechanism for pathology spreading in age‐related synucleinopathies, such as Parkinson's disease. In this study, the effects of overexpression‐induced α‐synuclein transfer were assessed over a 1‐year period after injection of viral vectors carrying human α‐synuclein DNA into the rat vagus nerve. This treatment causes targeted overexpression within neurons in the dorsal medulla oblongata and subsequent diffusion of the exogenous protein toward more rostral brain regions. Protein advancement and accumulation in pontine, midbrain, and forebrain areas were contingent upon continuous overexpression, because death of transduced medullary neurons resulted in cessation of spreading. Lack of sustained spreading did not prevent the development of long‐lasting pathological changes. Particularly remarkable were findings in the locus coeruleus, a pontine nucleus with direct connections to the dorsal medulla oblongata and greatly affected by overexpression‐induced transfer in this model. Data revealed progressive degeneration of catecholaminergic neurons that proceeded long beyond the time of spreading cessation. Neuronal pathology in the locus coeruleus was accompanied by pronounced microglial activation and, at later times, astrocytosis. Interestingly, microglial activation was also featured in another region reached by α‐synuclein transfer, the central amygdala, even in the absence of frank neurodegeneration. Thus, overexpression‐induced spreading, even if temporary, causes long‐lasting pathological consequences in brain regions distant from the site of overexpression but anatomically connected to it. Neurodegeneration may be a consequence of severe protein burden, whereas even a milder α‐synuclein accumulation in tissues affected by protein transfer could induce sustained microglial activation.


Summary
Increased expression of a-synuclein can initiate its long-distance brain transfer, representing a potential mechanism for pathology spreading in age-related synucleinopathies, such as Parkinson's disease. In this study, the effects of overexpressioninduced a-synuclein transfer were assessed over a 1-year period after injection of viral vectors carrying human a-synuclein DNA into the rat vagus nerve. This treatment causes targeted overexpression within neurons in the dorsal medulla oblongata and subsequent diffusion of the exogenous protein toward more rostral brain regions. Protein advancement and accumulation in pontine, midbrain, and forebrain areas were contingent upon continuous overexpression, because death of transduced medullary neurons resulted in cessation of spreading. Lack of sustained spreading did not prevent the development of long-lasting pathological changes.
Particularly remarkable were findings in the locus coeruleus, a pontine nucleus with direct connections to the dorsal medulla oblongata and greatly affected by overexpression-induced transfer in this model. Data revealed progressive degeneration of catecholaminergic neurons that proceeded long beyond the time of spreading cessation. Neuronal pathology in the locus coeruleus was accompanied by pronounced microglial activation and, at later times, astrocytosis. Interestingly, microglial activation was also featured in another region reached by a-synuclein transfer, the central amygdala, even in the absence of frank neurodegeneration. Thus, overexpressioninduced spreading, even if temporary, causes long-lasting pathological consequences in brain regions distant from the site of overexpression but anatomically connected to it. Neurodegeneration may be a consequence of severe protein burden, whereas even a milder a-synuclein accumulation in tissues affected by protein transfer could induce sustained microglial activation.

K E Y W O R D S
axon, locus coeruleus, microglia, neurodegeneration, Parkinson's disease, stereology 1 | INTRODUCTION mutation, alanine to threonine, at position 53 (A53T) of the a-synuclein gene (SNCA), and the development of autosomal-dominant parkinsonism (Polymeropoulos et al., 1997). Further genetic studies identified additional SNCA missense mutations (A30P, E46K, H50Q, G51D, and A53E) linked to hereditary parkinsonism (reviewed in Petrucci, Ginevrino & Valente, 2016). They also revealed an intriguing association between familial parkinsonism and SNCA duplication and triplication (Chartier-Harlin et al., 2004;Ib añez et al., 2004;Singleton et al., 2003). In patients with these multiplication mutations, the severity of clinical presentation is correlated with gene dosage, because triplication carriers display more severe symptoms and earlier disease onset than patients with SNCA duplication (Ross et al., 2008). Pathological features triggered by SNCA multiplication are similar to those seen in idiopathic Parkinson's disease, including the degeneration of specific neuronal populations and the accumulation of a-synuclein-containing intraneuronal inclusions (Konno, Ross, Puschmann, Dickson & Wszolek, 2016). Taken together, these observations indicate that a sustained increase in protein expression is itself capable of triggering a gain of a-synuclein toxic function, leading to development of Parkinson's disease-like pathology. They also support the possibility that even transient and more localized elevations of brain levels of a-synuclein could have deleterious effects and contribute to the development of synucleinopathies .
Studies over the past few years have characterized an a-synuclein property of likely relevance to its pathological role in human diseases: monomeric and aggregated a-synuclein are able to pass across neurons from donor to recipient cells and can thus advance from brain region to brain region following anatomical connections (Desplats et al., 2009;Goedert, Spillantini, Del Tredici & Braak, 2013;Rey, Petit, Bousset, Melki & Brundin, 2013). In Parkinson's disease, this interneuronal protein transfer may account for the stereotypical pattern of progression of a-synuclein pathology that, starting in the lower brainstem, affects higher and higher brain regions and ultimately reaches cortical areas Goedert et al., 2013). Recent experimental work has demonstrated that brain spreading of a-synuclein can be initiated by its overexpression.
Caudo-rostral protein transmission from the lower brainstem to the forebrain was prompted by targeted a-synuclein overexpression within neurons of the rat or mouse medulla oblongata (Helwig et al., 2016;. Experimental evidence also revealed long-distance a-synuclein diffusion from the brain to peripheral tissues (i.e. the stomach wall) as a result of enhanced protein expression in the rat midbrain (Ulusoy et al., 2017). Thus, a pathogenetic mechanism by which increased protein load could promote the development and progression of a synucleinopathy may be via interand intraneuronal transmission of a-synuclein and a-synuclein species (e.g. oligomers) with toxic potential (Helwig et al., 2016;Kim et al., 2013;Rochenstain et al., 2014).
Age-related synucleinopathies are characterized by a chronic disease course and progressive pathology, underscoring the importance of investigations into the long-term consequences of neuron-to-neuron transmission and widespread diffusion of a-synuclein. In this study, a-synuclein spreading triggered by its overexpression in the rat medulla oblongata was assessed over a period of 1 year, significantly extending the observation time of earlier investigations, and was correlated with short-and long-term markers of tissue injury.
The new findings elucidate important factors that affect in vivo asynuclein transfer and its pathological outcomes under conditions of enhanced protein load. Data reveal for the first time that long-term consequences of overexpression-induced spreading include neurodegenerative changes and robust microglial and astrocytic reactions in tissues affected by "secondary" (i.e. post-transfer) a-synuclein burden.  2.2 | Overexpression-induced spreading of asynuclein Overexpression of ha-synuclein triggers its neuron-to-neuron transmission from medullary donor neurons into recipient axons reaching the dorsal medulla oblongata from higher brain regions; through these axons, ha-synuclein then spreads toward the pons, midbrain, and forebrain (Helwig et al., 2016;. The next set of analyses was designed to assess how death of donor neurons at later times post-transduction affected ha-synuclein brain propagation. Spreading was estimated by counting the number of axons immunoreactive for ha-synuclein in brain sections at predetermined had spread from the medulla oblongata first to the pons and caudal midbrain (by 6 weeks) and then to the rostral midbrain and forebrain. No further advancement occurred thereafter and, in fact, a pronounced decrease in counts of ha-synuclein-positive fibers was found in all brain regions at 6 months and 1 year; at these later time points, positive axons became sparse in the pons, rare in the caudal midbrain and virtually undetectable in the rostral midbrain and forebrain ( Figure 2a). Axonal counts obtained in syn211-stained sections were validated in a separate set of samples labeled with a polyclonal antibody recognizing both human and rodent a-synuclein (AB5038P).
The pattern and extent of protein spreading were similar between syn211-and AB5038P-stained samples, and, in particular, data confirmed a dramatic loss of immunoreactive fibers between 3 and 6 months after AAV administration ( Figure 2b).

a-synuclein spreading
Spreading of ha-synuclein and its consequent accumulation within recipient neurons were accompanied by morphological evidence of axonal pathology; syn211-or AB5038P-labeled fibers appeared as tortuous threads with irregularly spaced and intensely labeled swellings ( Figure 2c and Figure S1). The volume of these swellings is an indicator of ha-synuclein burden . Mean volumes of axonal varicosities were compared in pontine sections of AAVinjected rats at different time points post-treatment. Interestingly, they varied significantly, reaching their peak at 3 months and then declining by 25% at 6 months and 50% at 1 year ( Figure 2c,d). Further characterization of axonal pathology was carried out using antibodies that recognize modified or aggregated forms of a-synuclein.
Phosphorylation at serine 129 is often used as a marker of a-synuclein pathology (Neumann et al., 2002). However, when pontine, midbrain, and forebrain sections from AAV-injected rats were stained for phospho-Ser129 a-synuclein, no immunoreactivity was detected at any time point post-treatment (data not shown). To assess protein aggregation, tissues were labeled with two conformation-specific antibodies: Syn-O2 recognizes both early (oligomers) and late (fibrils) a-synuclein aggregates, whereas Syn-F1 specifically reacts against fibrillar a-synuclein (Helwig et al., 2016;Vaikath et al., 2015).
Immunoreactivity could be seen in sections labeled with Syn-O2 but Astrogliosis is another typical reaction to brain tissue injury and was therefore evaluated as a potential pathological consequence of ha-synuclein spreading and ha-synuclein-induced neurodegeneration.
The number of astrocytes immunoreactive for glial fibrillary acidic protein (GFAP) was first estimated in the locus coeruleus of na€ ıve rats. Similar to the effect of aging on microglia, astrocyte counts were found to be elevated by 40% and 75% at 6 months and 1 year, respectively, as compared to earlier time points (Figure 3f). AAV represents a primary site of pathological a-synuclein accumulation (Braak, R€ ub, Gai &. Pathological features of both the locus coeruleus and central amygdala in Parkinson's disease brain also include neuronal cell loss, underscoring the relevance of these two regions for studies on the relationship between a-synuclein pathology and neurodegeneration (German et al., 1992;Harding, Stimson, Henderson & Halliday, 2002). The locus coeruleus and central amygdala densely project to the DMnX and, for this reason, may represent preferential sites of caudo-rostral a-synuclein spreading in Parkinson's disease as well as in our present model of overexpression-induced protein transmission . In this model, ha-synuclein burden in brain regions affected by protein spreading is inversely correlated with their distance from the medulla oblongata and, indeed, the number of ha-synucleinloaded axons and the extent of axonal protein accumulation are much greater in the locus coeruleus (pons) than in the amygdala (forebrain) . This difference allowed us to assess whether potential long-term tissue injury was dependent upon severity of the initial a-synuclein burden.
Findings of this study provide first experimental evidence linking overexpression-induced a-synuclein spreading to neurodegeneration. or hypertrophic (f) microglia was counted stereologically in the left central amygdala of age-matched na€ ıve controls (black bars) and AAVinjected (gray bars) rats. Error bars indicate SEM. *p < .05 vs. the value in age-matched controls at the corresponding time point (Student's t test). + p < .05 vs. the value at the earlier time point in the respective treatment group (age-matched controls or AAV-injected rats) (one-way ANOVA). # p < .05 vs. the value at the 3-month time point in the respective treatment group (one-way ANOVA) warranted to rule out the possibility, for example, that a more discrete protein transfer might have been overlooked due to limitations of the detection methods used in our present investigation.
Findings in the locus coeruleus were compared to results of parallel analyses in the central amygdala where, as discussed above, the extent of ha-synuclein spreading was significantly less pronounced. No evidence of neurodegeneration was found in the central amygdala, thus suggesting that a less severe ha-synuclein burden is compatible with clearance of the overloaded protein and neuronal survival. Less severe forebrain injury is also likely to explain the lack of astrocytic reaction in the central amygdala of AAV-injected rats even at late time points post-treatment. Quite in contrast, an intriguing long-term effect of ha-synuclein spreading in the central amygdala was an increase in counts of IBA1positive cells at 6 months and 1 year. Taken together, data indicate that astrogliosis is more strictly associated with neurodegenerative changes while activation of microglia can still occur in the absence of severe tissue injury; data are also consistent with the possibility that milder axonal pathology may facilitate the release of ha-synuclein into the extracellular space and its consequent binding to microglial TLR2. As compared to findings in the locus coeruleus where neuronal cell loss was associated with higher counts of hypertrophic and amoeboid microglia, changes in the central amygdala were accounted for by increases in hyper-ramified and hypertrophic cells. In line with earlier results (Sanchez-Guajardo, Febbraro, Kirik & Romero-Ramos, 2010), these observations suggest that microglial morphology is in part dependent upon the severity of a-synuclein-induced pathology; in particular, a marked elevation of IBA1-positive cells with amoeboid phenotype appears to be a reflection of pronounced tissue injury leading to neurodegeneration.
In summary, results of this study have elucidated a number of important factors and mechanisms affecting overexpressioninduced a-synuclein spreading and pathology. They include the survival/demise of donor neurons, the extent of protein burden within recipient cells, and the response of brain tissue to a-synuclein accumulation/release. A potential addition to this list is suggested by our findings in untreated na€ ıve animals. Neuronal counts were similar among na€ ıve rats of different ages in either the locus coeruleus or central amygdala. Age-related and regionspecific differences were seen, however, in glial counts. In the locus coeruleus, the number of microglia and astrocytes was progressively higher at 6 months and 1 year, whereas, in the central amygdala, a slight but statistically significant elevation of microglia was detected only at 1 year. The pathophysiological relevance of glial changes in the aging brain and their involvement in the pathogenesis of human synucleinopathies are far from being fully understood (Askew et al., 2017;Cotrina & Nedergaard, 2002;Hefendehl et al., 2014). It is reasonable to speculate, however, that age-and region-dependent increases in astrocyte and microglial counts could play a role in modulating long-term a-synuclein pathology and may contribute to selective vulnerability to a-synuclein spreading.

| EXPERIMEN TAL PROCED URES
A detailed description of the experimental procedures can be found in the online supporting information (Appendix S1).

| Animals and surgical procedures
Experimental protocols/procedures were approved by the ethical committee of the State Agency for Nature, Environment and Consumer Protection in North Rhine Westphalia. Some of the animals received intravagal injections of recombinant AAVs (serotype 2 genome and serotype 6 capsids) for transgene expression of ha-synuclein. The surgical procedure for vagal AAV injection has been previously described .

| Tissue preparation and histology
Animals were killed under pentobarbital anesthesia and perfused through the ascending aorta with paraformaldehyde. Brains were removed, immersion-fixed in paraformaldehyde, and cryopreserved.
Coronal sections (40 lm) throughout the brain were cut and stored.

| Statistical analysis
Statistical analyses were performed with PRISM software (version 7.0a; GraphPad Software, La Jolla, CA, USA). For normally distributed data, means between two groups were compared with two-tailed Student's t test, and comparisons between multiple groups were carried out with one-way ANOVA followed by Tukey post hoc test. For non-normally distributed data, Kruskal-Wallis test was applied. Statistical significance was set at p < .05. The number of animals used for each experiment/analysis is indicated in Table S1. RUSCONI ET AL. | 9 of 11