Amanda N. Sacino

IL-10 Alters Immunoproteostasis in APP Mice, Increasing Plaque Burden and Worsening Cognitive Behavior

Anti-inflammatory strategies are proposed to have beneficial effects in Alzheimers disease. To explore how anti-inflammatory cytokine signaling affects Aβ pathology, we investigated the effects of adeno-associated virus (AAV2/1)-mediated expression of Interleukin (IL)-10 in the brains of APP transgenic mouse models. IL-10 expression resulted in increased Aβ accumulation and impaired memory in APP mice. A focused transcriptome analysis revealed changes consistent with enhanced IL-10 signaling and increased ApoE expression in IL-10-expressing APP mice. ApoE protein was selectively increased in the plaque-associated insoluble cellular fraction, likely because of direct interaction with aggregated Aβ in the IL-10-expressing APP mice. Ex vivo studies also show that IL-10 and ApoE can individually impair glial Aβ phagocytosis. Our observations that IL-10 has an unexpected negative effect on Aβ proteostasis and cognition in APP mouse models demonstrate the complex interplay between innate immunity and proteostasis in neurodegenerative diseases, an interaction we call immunoproteostasis.

Intramuscular injection of α-synuclein induces CNS α-synuclein pathology and a rapid-onset motor phenotype in transgenic mice

Significance α-Synuclein (αS) inclusions are a hallmark of many progressive neurodegenerative disorders. Previously, intracerebral injection of exogenous preformed fibrillar αS in mouse models was shown to induce neuronal αS aggregation—a finding that has been interpreted as a prion-like mechanism. We now show that αS inclusion pathology can be induced in the brain and spinal cord of αS transgenic mice by a single peripheral intramuscular injection of αS. The formation of αS inclusions occurred concurrently with the presentation of a motor impairment in mice expressing mutant Ala53Thr human αS. This new model of robust and predictable induction of αS pathology will be especially valuable to further study the pathogenic mechanisms and assessment of therapeutic interventions. It has been hypothesized that α-synuclein (αS) misfolding may begin in peripheral nerves and spread to the central nervous system (CNS), leading to Parkinson disease and related disorders. Although recent data suggest that αS pathology can spread within the mouse brain, there is no direct evidence for spread of disease from a peripheral site. In the present study, we show that hind limb intramuscular (IM) injection of αS can induce pathology in the CNS in the human Ala53Thr (M83) and wild-type (M20) αS transgenic (Tg) mouse models. Within 2–3 mo after IM injection in αS homozygous M83 Tg mice and 3–4 mo for hemizygous M83 Tg mice, these animals developed a rapid, synchronized, and predictable induction of widespread CNS αS inclusion pathology, accompanied by astrogliosis, microgliosis, and debilitating motor impairments. In M20 Tg mice, starting at 4 mo after IM injection, we observed αS inclusion pathology in the spinal cord, but motor function remained intact. Transection of the sciatic nerve in the M83 Tg mice significantly delayed the appearance of CNS pathology and motor symptoms, demonstrating the involvement of retrograde transport in inducing αS CNS inclusion pathology. Outside of scrapie-mediated prion disease, to our knowledge, this findiing is the first evidence that an entire neurodegenerative proteinopathy associated with a robust, lethal motor phenotype can be initiated by peripheral inoculation with a pathogenic protein. Furthermore, this facile, synchronized rapid-onset model of α-synucleinopathy will be highly valuable in testing disease-modifying therapies and dissecting the mechanism(s) that drive αS-induced neurodegeneration.

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.

Brain Injection of α-Synuclein Induces Multiple Proteinopathies, Gliosis, and a Neuronal Injury Marker

Intracerebral injection of amyloidogenic α-synuclein (αS) has been shown to induce αS pathology in the CNS of nontransgenic mice and αS transgenic mice, albeit with varying efficiencies. In this study, using wild-type human αS transgenic mice (line M20), we demonstrate that intracerebral injection of recombinant amyloidogenic or soluble αS induces extensive αS intracellular inclusion pathology that is associated with robust gliosis. Near the injection site, a significant portion of αS inclusions are detected in neurons but also in astrocytes and microglia. Aberrant induction of expression of the intermediate filament protein peripherin, which is associated with CNS neuronal injury, was also observed predominantly near the site of injection. In addition, many pSer129 αS-induced inclusions colocalize with the low-molecular-mass neurofilament subunit (NFL) or peripherin staining. αS inclusion pathology was also induced in brain regions distal from the injection site, predominantly in neurons. Unexpectedly, we also find prominent p62-immunoreactive, αS-, NFL-, and peripherin-negative inclusions. These findings provide evidence that exogenous αS challenge induces αS pathology but also results in the following: (1) a broader disruption of proteostasis; (2) glial activation; and (3) a marker of a neuronal injury response. Such data suggest that induction of αS pathology after exogenous seeding may involve multiple interdependent mechanisms.

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.

Conformational templating of α-synuclein aggregates in neuronal-glial cultures

BackgroundGenetic studies have established a causative role for α-synuclein (αS) in Parkinson’s disease (PD), and the presence of αS aggregates in the form of Lewy body (LB) and Lewy neurite (LN) protein inclusions are defining pathological features of PD. Recent data has established that extracellular αS aggregates can induce intracellular αS pathologies supporting the hypothesis that αS pathology can spread via a “prion-like” self-templating mechanism.ResultsHere we investigated the potential for conformational templating of αS intracellular aggregates by seeding using recombinant wild-type and PD-linked mutant (A53T and E46K) αS in primary mixed neuronal-glial cultures. We find that wild-type and A53T αS fibrils predominantly seed flame-like inclusions in both neurons and astrocytes of mixed primary cultures; whereas the structurally distinct E46K fibrils seed punctate, rounded inclusions. Notably, these differences in seeded inclusion formation in these cultures reflect differences in inclusion pathology seen in transgenic mice expressing the A53T or E46K αS mutants. We further show that the inclusion morphology is dictated primarily by the seed applied rather than the form of αS expressed. We also provide initial evidence that αS inclusion pathology can be passaged in primary astrocyte cultures.ConclusionThese studies establish for the first time that αS aggregation in cultured cells can occur by a morphological self-templating mechanism.

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

Does a prion-like mechanism play a major role in the apparent spread of α-synuclein pathology?

Parkinsons disease, the most common movement disorder, results in an insidious reduction for patients in quality of life and ability to function. A hallmark of Parkinsons disease is the brain accumulation of neuronal cytoplasmic inclusions comprised of the protein α-synuclein. The presence of α-synuclein brain aggregates is observed in several neurodegenerative diseases, including dementia with Lewy bodies and Lewy body variant of Alzheimers disease. These disorders, as a group, are termed synucleinopathies. Mounting evidence indicates that α-synuclein amyloid pathology may spread during disease progression by a prion-like (self-templating alteration in protein conformation) mechanism. Clear in vitro and cell culture data demonstrate that amyloidogenic α-synuclein can readily induce the conversion of other α-synuclein molecules into this conformation. Some data from experimental mouse studies and autopsied brain analyses also are consistent with the notion that a self-promoting process of α-synuclein amyloid inclusion formation may lead to a progressive spread of disease in vivo. However, as pointed out in this review, there are alternative explanations and interpretations for these findings. Therefore, from a therapeutic perspective, it is critical to determine the relative importance and contribution of α-synuclein prionlike spread in disease before embarking on elaborate efforts to target this putative pathogenic mechanism.

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.