Mieu Brooks

Amyloidogenic α-synuclein seeds do not invariably induce rapid, widespread pathology in mice

In order to further evaluate the parameters whereby intracerebral administration of recombinant α-synuclein (αS) induces pathological phenotypes in mice, we conducted a series of studies where αS fibrils were injected into the brains of M83 (A53T) and M47 (E46K) αS transgenic (Tg) mice, and non-transgenic (nTg) mice. Using multiple markers to assess αS inclusion formation, we find that injected fibrillar human αS induced widespread cerebral αS inclusion formation in the M83 Tg mice, but in both nTg and M47 Tg mice, induced αS inclusion pathology is largely restricted to the site of injection. Furthermore, mouse αS fibrils injected into nTg mice brains also resulted in inclusion pathology restricted to the site of injection with no evidence for spread. We find no compelling evidence for extensive spread of αS pathology within white matter tracts, and we attribute previous reports of white matter tract spreading to cross-reactivity of the αS pSer129/81A antibody with phosphorylated neurofilament subunit L. These studies suggest that, with the exception of the M83 Tg mice which appear to be uniquely susceptible to induction of inclusion pathology by exogenous forms of αS, there are significant barriers in mice to widespread induction of αS pathology following intracerebral administration of amyloidogenic αS.

Induction of CNS α-synuclein pathology by fibrillar and non-amyloidogenic recombinant α-synuclein.

Backgroundα-Synuclein (αS) is the major component of several types of brain inclusions including Lewy bodies, a hallmark of Parkinson’s disease. Aberrant aggregation of αS also is associated with cellular demise in multiple neurologic disorders collectively referred to as synucleinopathies. Recent studies demonstrate the induction of αS pathology by a single intracerebral injection of exogenous amyloidogenic αS in adult non-transgenic and transgenic mice expressing human αS. To further investigate the mechanism of pathology induction and evaluate an experimental paradigm with potential for higher throughput, we performed similar studies in neonatal mice injected with αS.ResultsIn non-transgenic mice, we observed limited induction of neuronal αS inclusions predominantly 8 months after brain injection of aggregated, amyloidogenic human αS. More robust inclusion pathology was induced in transgenic mice expressing wild-type human αS (line M20), and inclusion pathology was observed at earlier time points. Injection of a non-amyloidogenic (Δ71-82) deletion protein of αS was also able to induce similar pathology in a subset of M20 transgenic mice. M20 transgenic mice injected with amyloidogenic or non-amyloidogenic αS demonstrated a delayed and robust induction of brain neuroinflammation that occurs in mice with or without αS pathological inclusions implicating this mechanism in aggregate formation.ConclusionsThe finding that a non-amyloidogenic Δ71-82 αS can induce pathology calls into question the simple interpretation that exogenous αS catalyzes aggregation and spread of intracellular αS pathology solely through a nucleation dependent conformational templating mechanism. These results indicate that several mechanisms may act synergistically or independently to promote the spread of αS pathology.

Robust Central Nervous System Pathology in Transgenic Mice following Peripheral Injection of α-Synuclein Fibrils

ABSTRACT Misfolded α-synuclein (αS) is hypothesized to spread throughout the central nervous system (CNS) by neuronal connectivity leading to widespread pathology. Increasing evidence indicates that it also has the potential to invade the CNS via peripheral nerves in a prion-like manner. On the basis of the effectiveness following peripheral routes of prion administration, we extend our previous studies of CNS neuroinvasion in M83 αS transgenic mice following hind limb muscle (intramuscular [i.m.]) injection of αS fibrils by comparing various peripheral sites of inoculations with different αS protein preparations. Following intravenous injection in the tail veins of homozygous M83 transgenic (M83+/+) mice, robust αS pathology was observed in the CNS without the development of motor impairments within the time frame examined. Intraperitoneal (i.p.) injections of αS fibrils in hemizygous M83 transgenic (M83+/−) mice resulted in CNS αS pathology associated with paralysis. Interestingly, injection with soluble, nonaggregated αS resulted in paralysis and pathology in only a subset of mice, whereas soluble Δ71-82 αS, human βS, and keyhole limpet hemocyanin (KLH) control proteins induced no symptoms or pathology. Intraperitoneal injection of αS fibrils also induced CNS αS pathology in another αS transgenic mouse line (M20), albeit less robustly in these mice. In comparison, i.m. injection of αS fibrils was more efficient in inducing CNS αS pathology in M83 mice than i.p. or tail vein injections. Furthermore, i.m. injection of soluble, nonaggregated αS in M83+/− mice also induced paralysis and CNS αS pathology, although less efficiently. These results further demonstrate the prion-like characteristics of αS and reveal its efficiency to invade the CNS via multiple routes of peripheral administration. IMPORTANCE The misfolding and accumulation of α-synuclein (αS) inclusions are found in a number of neurodegenerative disorders and is a hallmark feature of Parkinsons disease (PD) and PD-related diseases. Similar characteristics have been observed between the infectious prion protein and αS, including its ability to spread from the peripheral nervous system and along neuroanatomical tracts within the central nervous system. In this study, we extend our previous results and investigate the efficiency of intravenous (i.v.), intraperitoneal (i.p.), and intramuscular (i.m.) routes of injection of αS fibrils and other protein controls. Our data reveal that injection of αS fibrils via these peripheral routes in αS-overexpressing mice are capable of inducing a robust αS pathology and in some cases cause paralysis. Furthermore, soluble, nonaggregated αS also induced αS pathology, albeit with much less efficiency. These findings further support and extend the idea of αS neuroinvasion from peripheral exposures.

Proteolysis of α-synuclein fibrils in the lysosomal pathway limits induction of inclusion pathology.

Progression of α‐synuclein inclusion pathology may occur through cycles of release and uptake of α‐synuclein aggregates, which induce additional intracellular α‐synuclein inclusion pathology. This process may explain (i) the presence of α‐synuclein inclusion pathology in grafted cells in human brains, and (ii) the slowly progressive nature of most human α‐synucleinopathies. It also provides a rationale for therapeutic targeting of extracellular aggregates to limit pathology spread. We investigated the cellular mechanisms underlying intraneuronal α‐synuclein aggregation following exposure to exogenous preformed α‐synuclein amyloid fibrils. Exogenous α‐synuclein fibrils efficiently attached to cell membranes and were subsequently internalized and degraded within the endosomal/lysosomal system. However, internalized α‐synuclein amyloid fibrils can apparently overwhelm the endosomal/lysosomal machinery leading to the induction of intraneuronal α‐synuclein inclusions comprised of endogenous α‐synuclein. Furthermore, the efficiency of inclusion formation was relatively low in these studies compared to studies using primary neuronal‐glial cultures over‐expressing α‐synuclein. Our study indicates that under physiologic conditions, endosomal/lysosomal function acts as an endogenous barrier to the induction of α‐synuclein inclusion pathology, but when compromised, it may lower the threshold for pathology induction/transmission.

Non-prion-type transmission in A53T α-synuclein transgenic mice: a normal component of spinal homogenates from naïve non-transgenic mice induces robust α-synuclein pathology

identity of the inducing factor(s) in tissue homogenates has not been unequivocally resolved. To determine whether degenerating tissues might contain non-αS components that could induce pathology in M83+/− mice, we conducted brain injections of young M83+/− mice with spinal cord (SC) homogenates prepared from motor-impaired M83+/+ mice, motor-impaired transgenic mice expressing the G93A variant of human superoxide dismutase-1 (hSOD-1), and healthy non-transgenic (NTg) mice. The intracerebral (hippocampal) injection of SC homogenates from affected M83+/+ mice served as a positive control and these mice were sacrificed at 120 days post-injection (DPI), at which time αS pathology was predominantly observed in the forebrain (hippocampus and entorhinal cortex), but also distributed in the midbrain, brainstem, and SC (Supplemental Fig. 1; Table 1). For the intrahippocampal injections of SC homogenates from paralyzed G93A hSOD-1 and NTg mice, the study design was to sacrifice mice at 180 DPI to assess pathology levels. Unexpectedly, two of the M83+/− mice injected with SC homogenate from paralyzed G93A hSOD-1 mice developed motor impairments prior to 180 DPI, leading to complete hind limb paralysis (Table 1). These two mice and the other three asymptomatic mice, sacrificed at 180 DPI, all had prominent αS inclusion pathology with a distribution typical of aged M83 mice, with additional pathology observed in the hippocampus (Fig. 1; Table 1). Even more surprisingly, 4 out of the 5 M83+/− mice injected in the hippocampus with SC homogenates from NTg mice also developed M83-type motor impairment and paralysis (Table 1). M83+/− mice injected with either G93A hSOD-1 or NTg SC homogenates showed similar levels of αS inclusion pathology (Fig. 1; Table 1). One pre-symptomatic mouse from each of these cohorts was sacrificed at 105 DPI to survey for earlier pathologic induction and these also presented with αS inclusion The injection of tissue homogenates from diseased animals into naive animals to induce a neurodegenerative phenotype is a feature that defines “prion-like” transmission. Several recent studies have demonstrated that transgenic mice expressing human A53T-α-synuclein (αS; Line M83), respond to injections of CNS tissue homogenates containing abundant αS pathology by accelerated onset of CNS pathology with concurrent accelerated motor impairment [1, 3–5, 9]. Homozygous line M83+/+ A53T αS mice naturally develop a severe motor phenotype between 8 and 16 months that is associated with the formation of αS inclusions in the spinal cord, brain stem, thalamus, periaqueductal gray, and motor cortex [2]. Hemizygous M83+/− mice do not begin to develop these phenotypes until 21 months or later [2], but disease can be induced earlier by injection of CNS homogenates from affected M83+/+ mice [5, 9]. Although purified αS protein fibrils can reproduce the effects seen with tissue homogenates [1, 3, 6, 8], the

Novel antibodies to phosphorylated α-synuclein serine 129 and NFL serine 473 demonstrate the close molecular homology of these epitopes

Pathological inclusions containing aggregated, highly phosphorylated (at serine129) α-synuclein (αS pSer129) are characteristic of a group of neurodegenerative diseases termed synucleinopathies. Antibodies to the pSer129 epitope can be highly sensitive in detecting αS inclusions in human tissue and experimental models of synucleinopathies. However, the generation of extensively specific pSer129 antibodies has been problematic, in some cases leading to the misinterpretation of αS inclusion pathology. One common issue is cross-reactivity to the low molecular mass neurofilament subunit (NFL) phosphorylated at Ser473. Here, we generated a series of monoclonal antibodies to the pSer129 αS and pSer473 NFL epitopes. We determined the relative abilities of the known αS kinases, polo-like kinases (PLK) 1, 2 and 3 and casein kinase (CK) II in phosphorylating NFL and αS, while using this information to characterize the specificity of the new antibodies. NFL can be phosphorylated by PLK1, 2 and 3 at Ser473; however CKII shows the highest phosphorylation efficiency and specificity for this site. Conversely, PLK3 is the most efficient kinase at phosphorylating αS at Ser129, but there is overlay in the ability of these kinases to phosphorylate both epitopes. Antibody 4F8, generated to the pSer473 NFL epitope, was relatively specific for phosphorylated NFL, however it could uniquely cross-react with pSer129 αS when highly phosphorylated, further showing the structural similarity between these phospho-epitopes. All of the new pSer129 antibodies detected pathological αS inclusions in human brains and mouse and cultured cell experimental models of induced synucleinopathies. Several of these pSer129 αS antibodies reacted with the pSer473 NFL epitope, but 2 clones (LS3-2C2 and LS4-2G12) did not. However, LS3-2C2 demonstrated cross-reactivity with other proteins. Our findings further demonstrate the difficulties in generating specific pSer129 αS antibodies, but highlights that the use of multiple antibodies, such as those generated here, can provide a sensitive and accurate assessment of αS pathology.

Inefficient induction and spread of seeded tau pathology in P301L mouse model of tauopathy suggests inherent physiological barriers to transmission

Intercellular prion-like transmission of misfolded and aggregated tau protein along interconnected anatomic pathways is thought to contribute to the etiology of tauopathies, such as Alzheimer’s disease (AD) [4]. Indeed, the observation that synthetic tau fibrils and AD patient brain homogenates are capable of inducing robust tau pathology in tau transgenic mice strongly support the hypothesis that exogenous tau can trigger tauopathy by prion-like templating and transmission [1–3,6]. Transmission of tauopathy seems to depend on the molecular conformation of the exogenous seeds [8]. Therefore, characterizing the differential seeding and transmission properties of tau conformers will allow us to determine optimal therapeutics against tauopathies. Here, we evaluated the seeding and transmission properties of pre-fibrillized wild type K18 tau fragment which contains all four microtubule binding domains of the longest tau isoform and retains the amyloid fibril core [9]. Since sonication potentiates prion seeding [7], we investigated the seeding properties of K18 fibrils sonicated either 1) in a low-power water bath sonicator (“Bath”) yielding typical shorter fibrillar preparations or 2) with a metal probe sonicator (“Probe”) resulting predominantly in amorphous aggregates as observed by negative staining electron microscopy (Supplementary Figure 1). Using HEK293T cells overexpressing tau, we demonstrated that both types of sonicated K18 fibrils could efficiently induce intracellular detergent-insoluble tau inclusions in the presence of lipofectamine (Supplementary Figure 2). Next, we tested whether sonicated K18 fibrils induce tauopathy in homozygous JNPL3 tau transgenic mice expressing P301L tau (hP301L) (Supplementary Table 1) [5]. The protracted nature of disease progression makes the hP301L mice an excellent primed model to study seeding and transmission of tauopathy [5]. Sonicated K18 fibrils was bilaterally injected in the hippocampus of 2-month old hP301L mice and analyzed after 4 months. In female hP301L mice, probe-sonicated K18 fibrils, but not bath-sonicated fibrils, induced limited AT8 and PHF1 immunoreactive tau pathology directly at the injection site (hippocampus) and in the neuro-anatomically connected entorhinal cortex (arrows, Figure 1; Supplementary Figure 3). Tau pathology, typically observed in aged hP301L mice, was observed in the brainstem and midbrain of all mice, irrespective of injection. Male hP301L mice similarly injected in the hippocampus with probe-sonicated K18 fibrils failed to show any induction of tau pathology (Supplementary Figure 4). Since hP301L mice develop early spinal cord and brainstem tau pathology [5], we reasoned that if K18 fibrils trigger templated tau protein transmission depending on focal priming, injection of seeds in the gastrocnemius muscle (IM) or cisterna magna (ICM) might induce or potentiate tau pathology following projections along the spinal cord and brainstem. IM or ICM injection of sonicated K18 (water-bath and probe sonicated) fibrils did not result in increased tau pathology in the CNS, suggesting inefficient peripheral to central transmission of tauopathy (Supplementary Figures 5–6). Figure 1 Hippocampal injection of probe-sonicated K18 induces limited tauopathy in female hP301L mice. hP301L mice were bilaterally injected in the hippocampus (arrowhead, top panel) with K18 fibrils sonicated using a bath or probe sonicator, or PBS and analyzed ... In summary, we have demonstrated that though a) synthetic K18 aggregates display robust seeding in vitro, b) intra-hippocampal administration of probe-sonicated K18 fibrils lead to limited induction of tau pathology, and c) peripheral administration of K18 tau does not induce CNS tau pathology in hP301L mice. Our data suggests that significant physio-chemical barriers, dependent on factors such as physical proximity to the injection site and conformation of seeds used, regulates induction of tau pathology in hP301L mice. Our studies seemingly differ from a recent observation of robust tauopathy induction in P301L transgenic mice using K18/PL seeds [6] but these discrepancies may be explained by differences in the type (wild type K18 or K18/PL) and conformation of tau seeds used. Such intriguing observations raise further notions regarding differential transmission properties of tau conformers as well as the acceptor mouse model, ostensibly contributing to distinct physio-chemical properties of tau seeds as suggested by other groups [8]. Based on our data, we speculate that tau inclusion pathology follows a non-stochastic process where conformationally distinct misfolded tau fibrils with specific clinico-pathologic properties display differential seeding potential, perhaps leading to phenotypic diversity of tauopathies [8]. Overall, K18 seeding and spread of tau pathology is an inefficient process in the hP301L primed tau mouse model, highlighting the complex nature of intercellular transmission of tauopathy. These findings should stimulate further studies in harnessing the endogenous mechanisms that can modify or even impede the induction and transmission of tau pathology.

Prion-like transmission of α-synuclein pathology in the context of an NFL null background

Neurofilaments are a major component of the axonal cytoskeleton in neurons and have been implicated in a number of neurodegenerative diseases due to their presence within characteristic pathological inclusions. Their contributions to these diseases are not yet fully understood, but previous studies investigated the effects of ablating the obligate subunit of neurofilaments, low molecular mass neurofilament subunit (NFL), on disease phenotypes in transgenic mouse models of Alzheimers disease and tauopathy. Here, we tested the effects of ablating NFL in α-synuclein M83 transgenic mice expressing the human pathogenic A53T mutation, by breeding them onto an NFL null background. The induction and spread of α-synuclein inclusion pathology was triggered by the injection of preformed α-synuclein fibrils into the gastrocnemius muscle or hippocampus in M83 versus M83/NFL null mice. We observed no difference in the post-injection time to motor-impairment and paralysis endpoint or amount and distribution of α-synuclein inclusion pathology in the muscle injected M83 and M83/NFL null mice. Hippocampal injected M83/NFL null mice displayed subtle region-specific differences in the amount of α-synuclein inclusions however, pathology was observed in the same regions as the M83 mice. Overall, we observed only minor differences in the induction and transmission of α-synuclein pathology in these induced models of synucleinopathy in the presence or absence of NFL. This suggests that NFL and neurofilaments do not play a major role in influencing the induction and transmission of α-synuclein aggregation.

Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course

Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.

Erratum to: Amyloidogenic α-synuclein seeds do not invariably induce rapid, widespread pathology in mice

In our original paper, the rows of images in Fig. 1b were inverted and the bottom right (pSer129 stained) image was from the hypothalamic region and not the brainstem region as indicated in the figure legend. We have now correctly rearranged the rows of images to correspond with the text in the figure. We have also replaced the pSer129 immunostained image from the symptomatic, aging M83 mouse to reflect a region in the brainstem as indicated in the original legend.