Jacob I. Ayers

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.

Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases

Greater than 160 missense mutations in copper-zinc superoxide dismutase-1 (SOD1) can cause amyotrophic lateral sclerosis (ALS). These mutations produce conformational changes that reveal novel antibody binding epitopes. A monoclonal antibody, clone C4F6 - raised against the ALS variant G93A of SOD1, has been identified as specifically recognizing a conformation shared by many ALS mutants of SOD1. Attempts to determine whether non-mutant SOD1 adopts a C4F6-reactive conformation in spinal tissues of sporadic ALS (sALS) patients has produced inconsistent results. To define the epitope recognized by C4F6, we tested its binding to a panel of recombinant ALS-SOD1 proteins expressed in cultured cells, producing data to suggest that the C4F6 epitope minimally contains amino acids 90–93, which are normally folded into a tight hairpin loop. Multiple van der Waals interactions between the 90–93 loop and a loop formed by amino acids 37–42, particularly a leucine at position 38, form a stable structure termed the β-plug. Based on published modeling predictions, we suggest that the binding of C4F6 to multiple ALS mutants of SOD1 occurs when the local structure within the β-plug, including the loop at 90–93, is destabilized. In using the antibody to stain tissues from transgenic mice or humans, the specificity of the antibody for ALS mutant SOD1 was influenced by antigen retrieval protocols. Using conditions that showed the best discrimination between normal and misfolded mutant SOD1 in cell and mouse models, we could find no obvious difference in C4F6 reactivity to spinal motor neurons between sALS and controls tissues.

Widespread and Efficient Transduction of Spinal Cord and Brain Following Neonatal AAV Injection and Potential Disease Modifying Effect in ALS Mice

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.

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.

Distinct conformers of transmissible misfolded SOD1 distinguish human SOD1-FALS from other forms of familial and sporadic ALS

Evidence of misfolded wild-type superoxide dismutase 1 (SOD1) has been detected in spinal cords of sporadic ALS (sALS) patients, suggesting an etiological relationship to SOD1-associated familial ALS (fALS). Given that there are currently a number of promising therapies under development that target SOD1, it is of critical importance to better understand the role of misfolded SOD1 in sALS. We previously demonstrated the permissiveness of the G85R-SOD1:YFP mouse model for MND induction following injection with tissue homogenates from paralyzed transgenic mice expressing SOD1 mutations. This prompted us to examine whether WT SOD1 can self-propagate misfolding of the G85R-SOD1:YFP protein akin to what has been observed with mutant SOD1. Using the G85R-SOD1:YFP mice, we demonstrate that misfolded conformers of recombinant WT SOD1, produced in vitro, induce MND with a distinct inclusion pathology. Furthermore, the distinct pathology remains upon successive passages in the G85R-SOD1:YFP mice, strongly supporting the notion for conformation-dependent templated propagation and SOD1 strains. To determine the presence of a similar misfolded WT SOD1 conformer in sALS tissue, we screened homogenates from patients diagnosed with sALS, fALS, and non-ALS disease in an organotypic spinal cord slice culture assay. Slice cultures from G85R-SOD1:YFP mice exposed to spinal homogenates from patients diagnosed with ALS caused by the A4V mutation in SOD1 developed robust inclusion pathology, whereas spinal homogenates from more than 30 sALS cases and various controls failed. These findings suggest that mutant SOD1 has prion-like attributes that do not extend to SOD1 in sALS tissues.

Distinctive features of the D101N and D101G variants of superoxide dismutase 1; two mutations that produce rapidly progressing motor neuron disease.

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis induce misfolding and aggregation of the protein with the inherent propensity of mutant SOD1 to aggregate generally correlating, with a few exceptions, to the duration of illness in patients with the same mutation. One notable exception was the D101N variant, which has been described as wild‐type‐like. The D101N mutation is associated with rapidly progressing motor neuron degeneration but shows a low propensity to aggregate. By assaying the kinetics of aggregation in a well‐characterized cultured cell model, we show that the D101N mutant is slower to initiate aggregation than the D101G mutant. In this cell system of protein over‐expression, both mutants were equally less able to acquire Zn than WT SOD1. In addition, both of these mutants were equivalently less able to fold into the trypsin‐resistant conformation that characterizes WT SOD1. A second major difference between the two mutants was that the D101N variant more efficiently formed a normal intramolecular disulfide bond. Overall, our findings demonstrate that the D101N and D101G variants exhibit clearly distinctive features, including a different rate of aggregation, and yet both are associated with rapidly progressing disease.

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

Direct and indirect mechanisms for wild-type SOD1 to enhance the toxicity of mutant SOD1 in bigenic transgenic mice

Co-expression of wild-type human superoxide dismutase 1 (WT-hSOD1) with ALS mutant hSOD1 accelerates disease onset relative to mice expressing only mutant protein. Here, we analyzed the effect of co-expressed WT-hSOD1 in two established mutant mouse models (L126Z and G37R), and a new model that expresses the first 102 amino acids of SOD1 with mutations at histidines 46, 48 and 63 to eliminate Cu binding (Cu-V103Z). A subset of Cu-V103Z mice developed paralysis between 500 and 730 days. Similar to mice expressing L126Z-SOD1, the spinal cords of this new model showed SOD1 immunoreactive fibrillar inclusions. Co-expression of WT-hSOD1 with Cu-V103Z SOD1 moderately accelerated the age to paralysis, similar in magnitude to WT/L126Z mice. In either combination of these bigenic mice, the severity of fibrillar inclusion pathology was diminished and unreactive to antibodies specific for the C terminus of WT protein. Co-expression of WT-hSOD1 fused to yellow fluorescent protein (WT-hSOD1:YFP) with G37R-hSOD1 produced earlier disease, and spinal cords of paralyzed bigenic mice showed YFP fluorescent inclusion-like structures. In bigenic L126Z/WT-hSOD1:YFP mice, disease was not accelerated and WT-hSOD1:YFP remained diffusely distributed. A combination of split luciferase complementation assays and affinity capture-binding assays demonstrated that soluble G37R-hSOD1 efficiently and tightly bound soluble WT-hSOD1, whereas soluble forms of the Cu-V103Z and L126Z variants demonstrated low affinity. These data indicate that WT-hSOD1 may indirectly augment the toxicity of mutant protein by competing for protective factors, but disease onset seems to be most accelerated when WT-hSOD1 interacts with mutant SOD1 and becomes misfolded.

Relationship between mutant Cu/Zn superoxide dismutase 1 maturation and inclusion formation in cell models.

A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis, is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with amyotrophic lateral sclerosis mutations is slower to mature, and thus perhaps vulnerable to off‐pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In this study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time stamp that distinguishes pre‐existing and newly made proteins. In cells that transiently over‐express the Ala 4 to Val variant of SOD1‐Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over‐expressing untagged A4V‐SOD1, we observed that immature forms of the protein, lacking a Cu co‐factor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V‐SOD1, including newly made protein, are prone to misfolding and aggregation.

Substantially elevating the levels of αB-crystallin in spinal motor neurons of mutant SOD1 mice does not significantly delay paralysis or attenuate mutant protein aggregation.

There has been great interest in enhancing endogenous protein maintenance pathways such as the heat‐shock chaperone response, as it is postulated that enhancing clearance of misfolded proteins could have beneficial disease modifying effects in amyotrophic lateral sclerosis and other neurodegenerative disorders. In cultured cell models of mutant SOD1 aggregation, co‐expression of αB‐crystallin (αB‐crys) has been shown to inhibit the formation of detergent‐insoluble forms of mutant protein. Here, we describe the generation of a new line of transgenic mice that express αB‐crys at > 6‐fold the normal level in spinal cord, with robust increases in immunoreactivity throughout the spinal cord grey matter and, specifically, in spinal motor neurons. Surprisingly, spinal cords of mice expressing αB‐crys alone contained 20% more motor neurons per section than littermate controls. Raising αB‐crys by these levels in mice transgenic for either G93A or L126Z mutant SOD1 had no effect on the age at which paralysis developed. In the G93A mice, which showed the most robust degree of motor neuron loss, the number of these cells declined by the same proportion as in mice expressing the mutant SOD1 alone. In paralyzed bigenic mice, the levels of detergent‐insoluble, misfolded, mutant SOD1 were similar to those of mice expressing mutant SOD1 alone. These findings indicate that raising the levels of αB‐crys in spinal motor neurons by 6‐fold does not produce the therapeutic effects predicted by cell culture models of mutant SOD1 aggregation.