Pathogenic tau recruits wild-type tau into brain inclusions and induces gut degeneration in transgenic SPAM mice.

Pathological tau inclusions are neuropathologic hallmarks of many neurodegenerative diseases. We generated and characterized a transgenic mouse model expressing pathogenic human tau with S320F and P301S aggregating mutations (SPAM) at transgene levels below endogenous mouse tau protein levels. This mouse model develops a predictable temporal progression of tau pathology in the brain with biochemical and ultrastructural properties akin to authentic tau inclusions. Surprisingly, pathogenic human tau extensively recruited endogenous mouse tau into insoluble aggregates. Despite the early onset and rapid progressive nature of tau pathology, major neuroinflammatory and transcriptional changes were only detectable at later time points. Moreover, tau SPAM mice are the first model to develop loss of enteric neurons due to tau accumulation resulting in a lethal phenotype. With moderate transgene expression, rapidly progressing tau pathology, and a highly predictable lethal phenotype, the tau SPAM model reveals new associations of tau neurotoxicity in the brain and intestinal tract.

Novel monoclonal antibodies targeting the RRM2 domain of human TDP-43 protein.

Transactive response DNA-binding protein of 43 kilodaltons (TDP-43) is a 414 amino acid protein that under physiologic conditions localizes to the nucleus and participates in the regulation of RNA metabolism through two RNA recognition motifs (RRM1 and RRM2). In neurodegenerative diseases, TDP-43 may become hyperphosphorylated, ubiquitinated, and aggregate into cytoplasmic inclusions. TDP-43 is now well-characterized as a pathologic protein of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP). Additionally, a common TDP-43 proteinopathy arising outside of the context of ALS and FTLD-TDP has been recently described, termed "limbic predominant age-related TDP-43 encephalopathy (LATE)." In the current study, two novel mouse-derived monoclonal antibodies, 2G11 and 2H1, raised against an epitope within the RRM2 domain of TDP-43 (residues 198-216), were characterized for specificity and immunohistochemical application in human brain from cases of Alzheimer's disease (AD), Lewy Body Disease (LBD), amyotrophic lateral sclerosis (ALS), and frontotemporal lobe degeneration with TDP-43 inclusions (FTLD-TDP). Immunoblot analysis of these antibodies in HEK293T cells revealed efficient detection of intact human TDP-43 protein, and in N2A cells showed no reactivity for mouse TDP-43. Immunohistochemically applied to formalin-fixed paraffin-embedded tissues, 2G11 and 2H1 robustly identified the classic inclusions of ALS and FTLD-TDP, and efficaciously provided a diagnosis of LATE in cases of AD and LBD. These novel antibodies label aberrant intracytoplasmic protein inclusions without relying on hyperphosphorylated epitopes, and provide elegant discrimination between TDP-43 and tau neurofibrillary tangles within neurodegenerative comorbidity.

α-Synuclein Induces Progressive Changes in Brain Microstructure and Sensory-Evoked Brain Function That Precedes Locomotor Decline.

In vivo functional and structural brain imaging of synucleinopathies in humans have provided a rich new understanding of the affected networks across the cortex and subcortex. Despite this progress, the temporal relationship between α-synuclein (α-syn) pathology and the functional and structural changes occurring in the brain is not well understood. Here, we examine the temporal relationship between locomotor ability, brain microstructure, functional brain activity, and α-syn pathology by longitudinally conducting rotarod, diffusion magnetic resonance imaging (MRI), resting-state functional MRI (fMRI), and sensory-evoked fMRI on 20 mice injected with α-syn fibrils and 20 PBS-injected mice at three timepoints (10 males and 10 females per group). Intramuscular injection of α-syn fibrils in the hindlimb of M83+/- mice leads to progressive α-syn pathology along the spinal cord, brainstem, and midbrain by 16 weeks post-injection. Our results suggest that peripheral injection of α-syn has acute systemic effects on the central nervous system such that structural and resting-state functional activity changes occur in the brain by four weeks post-injection, well before α-syn pathology reaches the brain. At 12 weeks post-injection, a separate and distinct pattern of structural and sensory-evoked functional brain activity changes was observed that are co-localized with previously reported regions of α-syn pathology and immune activation. Microstructural changes in the pons at 12 weeks post-injection were found to predict survival time and preceded measurable locomotor deficits. This study provides preliminary evidence for diffusion and fMRI markers linked to the progression of synuclein pathology and has translational importance for understanding synucleinopathies in humans.SIGNIFICANCE STATEMENT α-Synuclein (α-syn) pathology plays a critical role in neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The longitudinal effects of α-syn pathology on locomotion, brain microstructure, and functional brain activity are not well understood. Using high field imaging, we show preliminary evidence that peripheral injection of α-syn fibrils induces unique patterns of functional and structural changes that occur at different temporal stages of α-syn pathology progression. Our results challenge existing assumptions that α-syn pathology must precede changes in brain structure and function. Additionally, we show preliminary evidence that diffusion and functional magnetic resonance imaging (fMRI) are capable of resolving such changes and thus should be explored further as markers of disease progression.

Carboxy-terminal truncation and phosphorylation of α-synuclein elongates survival in a prion-like seeding mouse model of synucleinopathy.

Pathologic intracellular inclusions formed from polymers of misfolded α-synuclein (αsyn) protein define a group of neurodegenerative diseases termed synucleinopathies which includes Parkinson's disease (PD). Prion-like recruitment of endogenous cellular αsyn has been demonstrated to occur in animal models of synucleinopathy, whereby misfolded αsyn can induce further pathologic αsyn inclusions to form through a prion-like mechanism. It has been suggested that misfolded αsyn may assume differing conformations which lead to varied clinical and pathological manifestations of disease; this phenomenon bears similarities to that of prion strains whereby the same misfolded protein can produce unique diseases. It is unclear what factors influence the development of unique αsyn strains, however post-translational modifications (PTMs) such as phosphorylation and truncation that are present in misfolded αsyn in disease may play a role due to their modulation of biochemical and structural αsyn properties. Herein, we investigate the prion-like properties of misfolded αsyn polymers containing either phosphomimetic (S129E) αsyn, 5 different major carboxy (C)-truncated forms of αsyn (1-115, 1-119, 1-122, 1-125, and 1-129 αsyn), or a mixture of these PTM containing αsyn forms compared to full-length (FL) αsyn in HEK293T cells and M83 transgenic mice overexpressing A53T αsyn. It is demonstrated that upon peripheral intramuscular injection of these C-truncated or S129E αsyn polymers into M83 mice, prion-like progression and time to disease onset in this mouse model is elongated when any of these PTMs are present, demonstrating that common modifications to the C-terminus of αsyn present in disease modulates the prion-like seeding properties of αsyn.

Generation and Characterization of Novel Monoclonal Antibodies Targeting p62/sequestosome-1 Across Human Neurodegenerative Diseases.

Human neurodegenerative diseases can be characterized as disorders of protein aggregation. As a key player in cellular autophagy and the ubiquitin proteasome system, p62 may represent an effective immunohistochemical target, as well as mechanistic operator, across neurodegenerative proteinopathies. In this study, 2 novel mouse-derived monoclonal antibodies 5G3 and 2A5 raised against residues 360-380 of human p62/sequestosome-1 were characterized via immunohistochemical application upon human tissues derived from cases of C9orf72-expansion spectrum diseases, Alzheimer disease, progressive supranuclear palsy, Lewy body disease, and multiple system atrophy. 5G3 and 2A5 reliably highlighted neuronal dipeptide repeat, tau, and α-synuclein inclusions in a distribution similar to a polyclonal antibody to p62, phospho-tau antibodies 7F2 and AT8, and phospho-α-synuclein antibody 81A. However, antibodies 5G3 and 2A5 consistently stained less neuropil structures, such as tau neuropil threads and Lewy neurites, while 2A5 marked fewer glial inclusions in progressive supranuclear palsy. Both 5G3 and 2A5 revealed incidental astrocytic tau immunoreactivity in cases of Alzheimer disease and Lewy body disease with resolution superior to 7F2. Through their unique ability to highlight specific types of pathological deposits in neurodegenerative brain tissue, these novel monoclonal p62 antibodies may provide utility in both research and diagnostic efforts.

Impaired tau-microtubule interactions are prevalent among pathogenic tau variants arising from missense mutations.

tau is a microtubule (MT)-associated protein that promotes tubulin assembly and stabilizes MTs by binding longitudinally along the MT surface. tau can aberrantly aggregate into pathological inclusions that define Alzheimer's disease, frontotemporal dementias, and other tauopathies. A spectrum of missense mutations in the tau-encoding gene microtubule-associated protein tau (MAPT) can cause frontotemporal dementias. tau aggregation is postulated to spread by a prion-like mechanism. Using a cell-based inclusion seeding assay, we recently reported that only a few tau variants are intrinsically prone to this type of aggregation. Here, we extended these studies to additional tau mutants and investigated their MT binding properties in mammalian cell-based assays. A limited number of tau variants exhibited modest aggregation propensity in vivo, but most tau mutants did not aggregate. Reduced MT binding appeared to be the most common dysfunction for the majority of tau variants due to missense mutations, implying that MT-targeting therapies could potentially be effective in the management of tauopathies.

Unique α-synuclein pathology within the amygdala in Lewy body dementia: implications for disease initiation and progression.

The protein α-synuclein (αsyn) forms pathologic aggregates in a number of neurodegenerative diseases including Lewy body dementia (LBD) and Parkinson's disease (PD). It is unclear why diseases such as LBD may develop widespread αsyn pathology, while in Alzheimer's disease with amygdala restricted Lewy bodies (AD/ALB) the αsyn aggregates remain localized. The amygdala contains αsyn aggregates in both LBD and in AD/ALB; to understand why αsyn pathology continues to progress in LBD but not in AD/ALB, tissue from the amygdala and other regions were obtained from 14 cases of LBD, 9 cases of AD/ALB, and 4 controls for immunohistochemical and biochemical characterization. Utilizing a panel of previously characterized αsyn antibodies, numerous unique pathologies differentiating LBD and AD/ALB were revealed; particularly the presence of dense neuropil αsyn aggregates, astrocytic αsyn, and αsyn-containing dystrophic neurites within senile plaques. Within LBD, these unique pathologies were predominantly present within the amygdala. Biochemically, the amygdala in LBD prominently contained specific carboxy-truncated forms of αsyn which are highly prone to aggregate, suggesting that the amygdala may be prone to initiate development of αsyn pathology. Similar to carboxy-truncated αsyn, it was demonstrated herein that the presence of aggregation prone A53T αsyn is sufficient to drive misfolding of wild-type αsyn in human disease. Overall, this study identifies within the amygdala in LBD the presence of unique strain-like variation in αsyn pathology that may be a determinant of disease progression.

Combining P301L and S320F tau variants produces a novel accelerated model of tauopathy.

Understanding the biological functions of tau variants can illuminate differential etiologies of Alzheimer's disease (AD) and primary tauopathies. Though the end-stage neuropathological attributes of AD and primary tauopathies are similar, the etiology and behavioral outcomes of these diseases follow unique and divergent trajectories. To study the divergent physiological properties of tau variants on a uniform immunogenetic background, we created somatic transgenesis CNS models of tauopathy utilizing neonatal delivery of adeno-associated viruses expressing wild-type (WT) or mutant tau in non-transgenic mice. We selected four different tau variants-WT tau associated with AD, P301L mutant tau associated with frontotemporal dementia (FTD), S320F mutant tau associated with Pick's disease and a combinatorial approach using P301L/S320F mutant tau. CNS-targeted expression of WT and P301L mutant tau results in robust tau hyperphosphorylation without tangle pathology, gradually developing age-progressive memory deficits. In contrast, the S320F variant, especially in combination with P301L, produces an AD-type tangle pathology, focal neuroinflammation and memory impairment on an accelerated time scale. Using the doubly mutated P301L/S320F tau variant, we demonstrate that combining different mutations can have an additive effect on neuropathologies and associated co-morbidities, possibly hinting at involvement of unique functional pathways. Importantly, we also show that overexpression of wild-type tau as well as an FTD-associated tau variant can lead to cognitive deficits even in the absence of tangles. Together, our data highlights the synergistic neuropathologies and associated cognitive and synaptic alterations of the combinatorial tau variant leading to a robust model of tauopathy.

Phosphorylation of serine 305 in tau inhibits aggregation.

Alzheimer's disease and other tauopathies are characterized by the brain accumulation of hyperphosphorylated aggregated tau protein forming pathological inclusions. Although elevated tau phosphorylated at many amino acid residues is a hallmark of pathological tau, some evidence suggest that tau phosphorylation at unique sites, especially within its microtubule-binding domain, might inhibit aggregation. In this study, the effects of phosphorylation of two unique residues within this domain, serine 305 (S305) and serine 320 (S320), were examined in the context of established aggregation and seeding models. It was found that the S305E phosphomimetic significantly inhibited both tau seeding and tau aggregation in this model, while S320E did not. To further explore S305 phosphorylation in vivo, a monoclonal antibody (2G2) specific for tau phosphorylated at S305 was generated and characterized. Consistent with inhibition of tau aggregation, phosphorylation of S305 was not detected in pathological tau inclusions in Alzheimer's disease brain tissue. This study indicates that phosphorylation of unique tau residues can be inhibitory to aggregate formation, and has important implications for potential kinase therapies. Additionally, it creates new tools for observing these changes in vivo.

Physiological C-terminal truncation of α-synuclein potentiates the prion-like formation of pathological inclusions.

α-Synuclein (αsyn) aggregates into toxic fibrils in multiple neurodegenerative diseases where these fibrils form characteristic pathological inclusions such as Lewy bodies (LBs). The mechanisms initiating αsyn aggregation into fibrils are unclear, but ubiquitous post-translational modifications of αsyn present in LBs may play a role. Specific C-terminally (C)-truncated forms of αsyn are present within human pathological inclusions and form under physiological conditions likely in lysosome-associated pathways, but the roles for these C-truncated forms of αsyn in inclusion formation and disease are not well understood. Herein, we characterized the in vitro aggregation properties, amyloid fibril structures, and ability to induce full-length (FL) αsyn aggregation through prion-like mechanisms for eight of the most common physiological C-truncated forms of αsyn (1-115, 1-119, 1-122, 1-124, 1-125, 1-129, 1-133, and 1-135). In vitro, C-truncated αsyn aggregated more readily than FL αsyn and formed fibrils with unique morphologies. The presence of C-truncated αsyn potentiated aggregation of FL αsyn in vitro through co-polymerization. Specific C-truncated forms of αsyn in cells also exacerbated seeded aggregation of αsyn. Furthermore, in primary neuronal cultures, co-polymers of C-truncated and FL αsyn were potent prion-like seeds, but polymers composed solely of the C-truncated protein were not. These experiments indicated that specific physiological C-truncated forms of αsyn have distinct aggregation properties, including the ability to modulate the prion-like aggregation and seeding activity of FL αsyn. Proteolytic formation of these C-truncated species may have an important role in both the initiation of αsyn pathological inclusions and further progression of disease with strain-like properties.

Motor neuron loss and neuroinflammation in a model of α-synuclein-induced neurodegeneration.

Mechanisms underlying α-synuclein (αSyn) mediated neurodegeneration are poorly understood. Intramuscular (IM) injection of αSyn fibrils in human A53T transgenic M83+/- mice produce a rapid model of α-synucleinopathy with highly predictable onset of motor impairment. Using varying doses of αSyn seeds, we show that αSyn-induced phenotype is largely dose-independent. We utilized the synchrony of this IM model to explore the temporal sequence of αSyn pathology, neurodegeneration and neuroinflammation. Longitudinal tracking showed that while motor neuron death and αSyn pathology occur within 2 months post IM, astrogliosis appears at a later timepoint, implying neuroinflammation is a consequence, rather than a trigger, in this prionoid model of synucleinopathy. Initiating at 3 months post IM, immune activation dominates the pathologic landscape in terminal IM-seeded M83+/- mice, as revealed by unbiased transcriptomic analyses. Our findings provide insights into the role of neuroinflammation in αSyn mediated proteostasis and neurodegeneration, which will be key in designing potential therapies.

Localized Induction of Wild-Type and Mutant Alpha-Synuclein Aggregation Reveals Propagation along Neuroanatomical Tracts.

Misfolded alpha-synuclein (αS) may exhibit a number of characteristics similar to those of the prion protein, including the apparent ability to spread along neuroanatomical connections. The demonstration for this mechanism of spread is largely based on the intracerebral injections of preaggregated αS seeds in mice, in which it cannot be excluded that diffuse, surgical perturbations and hematogenous spread also contribute to the propagation of pathology. For this reason, we have utilized the sciatic nerve as a route of injection to force the inoculum into the lumbar spinal cord and induce a localized site for the onset of αS inclusion pathology. Our results demonstrate that mouse αS fibrils (fibs) injected unilaterally in the sciatic nerve are efficient in inducing pathology and the onset of paralytic symptoms in both the M83 and M20 lines of αS transgenic mice. In addition, a spatiotemporal study of these injections revealed a predictable spread of pathology to brain regions whose axons synapse directly on ventral motor neurons in the spinal cord, strongly supporting axonal transport as a mechanism of spread of the αS inducing, or seeding, factor. We also revealed a relatively decreased efficiency for human αS fibs containing the E46K mutation to induce disease via this injection paradigm, supportive of recent studies demonstrating a diminished ability of this mutant αS to undergo aggregate induction. These results further demonstrate prion-like properties for αS by the ability for a progression and spread of αS inclusion pathology along neuroanatomical connections.IMPORTANCE The accumulation of alpha-synuclein (αS) inclusions is a hallmark feature of Parkinson's disease (PD) and PD-related diseases. Recently, a number of studies have demonstrated similarities between the prion protein and αS, including its ability to spread along neuroanatomical tracts throughout the central nervous system (CNS). However, there are caveats in each of these studies in which the injection routes used had the potential to result in a widespread dissemination of the αS-containing inocula, making it difficult to precisely define the mechanisms of spread. In this study, we assessed the spread of pathology following a localized induction of αS inclusions in the lumbar spinal cord following a unilateral injection in the sciatic nerve. Using this paradigm, we demonstrated the ability for αS inclusion spread and/or induction along neuroanatomical tracts within the CNS of two αS-overexpressing mouse models.

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.