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 Alzheimer's 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.

Comparison of the in vivo induction and transmission of α-synuclein pathology by mutant α-synuclein fibril seeds in transgenic mice.

Parkinson's disease (PD) is one of many neurodegenerative diseases termed synucleinopathies, neuropathologically defined by inclusions containing aggregated α-synuclein (αS). αS gene (SNCA) mutations can directly cause autosomal dominant PD. In vitro studies demonstrated that SNCA missense mutations may either enhance or diminish αS aggregation but cross-seeding of mutant and wild-type αS proteins appear to reduce aggregation efficiency. Here, we extended these studies by assessing the effects of seeded αS aggregation in αS transgenic mice through intracerebral or peripheral injection of various mutant αS fibrils. We observed modestly decreased time to paralysis in mice transgenic for human A53T αS (line M83) intramuscularly injected with H50Q, G51D or A53E αS fibrils relative to wild-type αS fibrils. Conversely, E46K αS fibril seeding was significantly delayed and less efficient in the same experimental paradigm. However, the amount and distribution of αS inclusions in the central nervous system were similar for all αS fibril muscle injected mice that developed paralysis. Mice transgenic for human αS (line M20) injected in the hippocampus with wild-type, H50Q, G51D or A53E αS fibrils displayed induction of αS inclusion pathology that increased and spread over time. By comparison, induction of αS aggregation following the intrahippocampal injection of E46K αS fibrils in M20 mice was much less efficient. These findings show that H50Q, G51D or A53E can efficiently cross-seed and induce αS pathology in vivo. In contrast, E46K αS fibrils are intrinsically inefficient at seeding αS inclusion pathology. Consistent with previous in vitro studies, E46K αS polymers are likely distinct aggregated conformers that may represent a unique prion-like strain of αS.

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

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 ßS, 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 Parkinson's 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.

Studies of lipopolysaccharide effects on the induction of α-synuclein pathology by exogenous fibrils in transgenic mice.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder that is pathologically characterized by loss of dopaminergic neurons from the substantia nigra, the presence of aggregated α-synuclein (αS) and evidence of neuroinflammation. Experimental studies have shown that the cerebral injection of recombinant fibrillar αS, especially in αS transgenic mouse models, can induce the formation and spread of αS inclusion pathology. However, studies reporting this phenomenon did not consider the presence of lipopolysaccharide (LPS) in the injected αS, produced in E. coli, as a potential confound. The objectives of this study are to develop a method to remove the LPS contamination and investigate the differences in pathologies induced by αS containing LPS or αS highly purified of LPS. RESULTS AND CONCLUSIONS: We were able to remove >99.5% of the LPS contamination from the αS preparations through the addition of a cation exchange step during purification. The αS pathology induced by injection of fibrils produced from αS containing LPS or purified of LPS, showed a similar distribution pattern; however, there was less spread into the cortex of the mice injected with αS containing higher levels of LPS. As previously reported, injection of αS fibrils could induce astrogliosis, and αS inclusions were present within astrocytes in mice injected with fibrils comprised of αS with or without cation exchange purification. Furthermore, we identified the presence of αS pathology in ependymal cells in both groups of mice, which suggests the involvement of a novel mechanism for spread in this model of αS pathology.