Fibrillar Inclusions Research Articles

TFE3-mediated neuroprotection: Clearance of aggregated α-synuclein and accumulated mitochondria in the AAV-α-synuclein model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions containing aggregated α-synuclein (α-Syn). While the pathology of PD is multifaceted, the aggregation of α-Syn and mitochondrial dysfunction are well-established hallmarks in its pathogenesis. Recently, TFE3, a transcription factor, has emerged as a regulator of autophagy and metabolic processes. However, it remains unclear whether TFE3 can facilitate the degradation of α-Syn and regulate mitochondrial metabolism specifically in dopaminergic neurons. In this study, we demonstrate that TFE3 overexpression significantly mitigates the loss of dopaminergic neurons and reduces the decline in tyrosine hydroxylase-positive fiber density, thereby restoring motor function in an α-Syn overexpression model of PD. Mechanistically, TFE3 overexpression reversed α-Syn-mediated impairment of autophagy, leading to enhanced α-Syn degradation and reduced aggregation. Additionally, TFE3 overexpression inhibited α-Syn propagation. TFE3 overexpression also reversed the down-regulation of Parkin, promoting the clearance of accumulated mitochondria, and restored the expression of PGC1-α and TFAM, thereby enhancing mitochondrial biogenesis in the adeno-associated virus-α-Syn model. These findings further underscore the neuroprotective role of TFE3 in PD and provide insights into its underlying mechanisms, suggesting TFE3 as a potential therapeutic target for PD.

Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

Multiple system atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.

The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study.

The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer's disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-β-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.

Cellular processing of α-synuclein fibrils results in distinct physiological C-terminal truncations with a major cleavage site at residue Glu 114

α-synuclein (αS) is an abundant, neuronal protein that assembles into fibrillar pathological inclusions in a spectrum of neurodegenerative diseases that include Lewy body diseases (LBD) and Multiple System Atrophy (MSA). The cellular and regional distributions of pathological inclusions vary widely between different synucleinopathies contributing to the spectrum of clinical presentations. Extensive cleavage within the carboxy (C)-terminal region of αS is associated with inclusion formation, although the events leading to these modifications and the implications for pathobiology are of ongoing study. αS preformed fibrils can induce prion-like spread of αS pathology in both invitro and animal models of disease. Using C truncation-specific antibodies, we demonstrated here that prion-like cellular uptake and processing of αS preformed fibrils resulted in two major cleavages at residues 103 and 114. A third cleavage product (122 αS) accumulated upon application of lysosomal protease inhibitors. Invitro, both 1-103 and 1-114 αS polymerized rapidly and extensively in isolation and in the presence of full-length αS. 1-103 αS also demonstrated more extensive aggregation when expressed in cultured cells. Furthermore, we used novel antibodies to αS cleaved at residue Glu114, to assess x-114 αS pathology in postmortem brain tissue from patients with LBD and MSA, as well as three different transgenic αS mouse models of prion-like induction. The distribution of x-114 αS pathology was distinct from that of overall αS pathology. These studies reveal the cellular formation and behavior of αS C-truncated at residues 114 and 103 as well as the disease dependent distribution of x-114 αS pathology.

Understanding the mechanism of fibrillization of hnRNPA1 from phase-separated condensates.

Stress granules are membrane-less cytosolic organelles that form through phase separation of RNA-binding proteins and translationally stalled mRNAs. Persistent stress granules have been implicated in the pathogenesis of neurodegenerative disorders such as amyotrophic lateral sclerosis and frontotemporal dementia, in which certain stress granule proteins form the hallmark fibrillar inclusions in affected cells. Heterogeneous nuclear ribonucleoprotein 1 (hnRNPA1) is one of these stress granule proteins, whose C-terminal low-complexity domain (LCD) is sufficient for mediating both phase separation and fibril formation. Disease mutations in the LCD enhance its fibril formation in vitro, and phase separation further enhances fibrillization, but the underlying mechanism linking the two processes is still poorly understood. Proposed mechanisms include enhanced nucleation from the dense phase and further enhancement by disease mutants if they have a higher driving force for phase separation. Here we find that disease mutants of hnRNPA1-LCD that enhance fibrillization show reduced driving force for phase-separation. A combination of bulk fluorescence and microscopy-based assays reveal that fibrillization is nucleated at the interface of condensates. Subsequent fibril growth, in contrast, is slowed down in the interior of WT hnRNPA1-LCD condensates, but not appreciably so in case of disease mutants. Our data supports a competition model between sticker-sticker interactions that drive phase separation and zipper-zipper interactions that drive fibril formation inside the dense phase environment. Stickers are uniformly distributed over the sequence and form frequent crosslinks, decreasing the probability of zippers to productively interact with each other, and thus weakening fibrillization. In contrast, the stronger zippers in disease mutants can effectively outcompete sticker interactions and enhance fibrillization. Our results suggest a potential protective role for phase separation in preventing fibril formation, whereas disease mutations turn stress granules into crucibles for fibrillization.

Structure and Pattern Formation in Biological Liquid Crystals: Insights From Theory and Simulation of Self-Assembly and Self-Organization

This review presents theory and simulation of liquid crystal phase ordering in biological fibrous materials, solutions, and composites in the presence of elastic fields, second phase inclusions, and transport phenomena, including complex shear-extensional flow and mass transfer. Liquid crystal self-assembly through phase ordering on elastic deformable membranes is first applied to characterize the mechanisms that control the structures in plant cell walls, highlighting how curvophobic and curvophilic effects introduce new structuring fields beyond hard-core repulsion. Then chiral nematic self-assembly is simulated in a mesophase containing fibrillar colloidal inclusions (liquid crystal-fibre composites) to demonstrate how the inclusion positional order generates defects and disclinations as shown in the plant cell wall. Coupling phase ordering to tuned transport phenomena is shown how and why it leads to self-organization such as paranematic states of dilute acidic aqueous collagen solutions. Further directed dehydration of well-organized paranematic collagen leads to defect free cholesteric films only when directed dehydration is synchronized with chirality formation. In addition, the ubiquitous surface nanowrinkling of cholesterics is captured with surface anchoring. In these four representative systems, the new mechanisms that enhance the well-known exclude volume interactions are identified quantified and validated with experimental data. Future directions to create new advanced multifunctional materials based on principles of self-assembly and self-organization are identified by leveraging the new couplings between material structure, geometry, and transport phenomena.

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer's disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope

BackgroundThe microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes.ResultsAs starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions.ConclusionsTau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts.

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

A thiol-based intramolecular redox switch in four-repeat tau controls fibril assembly and disassembly

Oxidative stress has been implicated in the pathogenesis and progression of several tauopathies, including Alzheimer's disease. The deposition of fibrillar inclusions made of tau protein is one of the pathological hallmarks of these disorders. Although it is becoming increasingly evident that the specific fibril structure may vary from one tauopathy to another and it is recognized that different types of isoforms (three-repeat and four-repeat tau) can be selectively deposited, little is known about the role oxidation may play in aggregation. Four-repeat tau contains two cysteines that can form an intramolecular disulfide bond, resulting in a structurally restrained compact monomer. There is discrepancy as to whether this monomer can aggregate or not. Using isolated four-repeat tau monomers (htau40) with intramolecular disulfide bonds, we demonstrate that these proteins form fibrils. The fibrils are less stable than fibrils formed under reducing conditions but are highly effective in seeding oxidized tau monomers. Conversely, a strong seeding barrier prevents incorporation of reduced tau monomers, tau mimics in which the cysteines have been replaced by alanines or serines, and three-repeat tau (htau23), a single-cysteine isoform. The barrier also holds true when seed and monomer types are reversed, indicating that oxidized and reduced tau are incompatible with each other. Surprisingly, fibrils composed of compact tau disaggregate upon reduction, highlighting the importance of the intramolecular disulfide bond for fibril stability. The findings uncover a novel binary redox switch that controls the aggregation and disaggregation of these fibrils and extend the conformational spectrum of tau aggregates.

3D fibrillated network of compatibilized linear low density polyethylene/polyamide with high melt strength and superior foamability

A new, efficient and easy to scale up approach to enhance the melt strength of linear low density polyethylene (LLDPE) through high aspect ratio polyamide (PA) fibrillar inclusions is presented. For the first time, the effect of the incorporation of compatibilizer in the blends on the efficiency of the in-situ fibrillation approach to improve rheological properties and foamability is discussed. The morphological analysis indicates high aspect ratio fibers were achieved for both compatibilized and non-compatibilized systems. Also, very fine fibers with nano-scale cross-sectional diameter were achieved for compatibilized blends. Comparable rheological responses and substantial improvement of the foaming ability with superior foam morphology and high cell density was obtained, for both fibrillated blends with and without compatibilizer. Investigation of the crystallinity of the blends through DSC and WAXD indicated that bimodal melting and crystallization of LLDPE is remarkably affected by the presence of PA inclusions, particularly for the blends containing fibrillated PA. Further investigation of the crystallinity through 2D-SAXS revealed formation of shish-kebab crystalline structures as a result of drawing and shear induced crystallization.

Stereotaxic Intracranial Delivery of Chemicals, Proteins or Viral Vectors to Study Parkinson's Disease.

Parkinson's disease (PD) is a progressive disorder traditionally defined by resting tremor and akinesia, primarily due to loss of dopaminergic neurons in the substantia nigra. Affected brain areas display intraneuronal fibrillar inclusions consisting mainly of alpha-synuclein (asyn) proteins. No animal model thus far has recapitulated all characteristics of this disease. Here, we describe the use of stereotaxic injection to deliver chemicals, proteins, or viral vectors intracranially in order to mimic various aspects of PD. These methods are well-established and widely used throughout the PD field. Stereotaxic injections are incredibly flexible; they can be adjusted in concentration, age of animal used for injection, brain area targeted and in animal species used. Combinations of substances allow for rapid variations to assess treatments or alter severity of the pathology or behavioral deficits. By injecting toxins into the brain, we can mimic inflammation and/or a severe loss of dopaminergic neurons resulting in substantial motor phenotypes. Viral vectors can be used to transduce cells to mimic genetic or mechanistic aspects. Preformed fibrillar asyn injections best recapitulate the progressive phenotype over an extended period of time. Once these methods are established, it can be economical to generate a new model compared to creating a new transgenic line. However, this method is labor intensive as it requires 30 minutes to four hours per animal depending on the model used. Each animal will have a slightly different targeting and therefore create a diverse cohort which on one hand can be challenging to interpret results from; on the other hand, help mimic a more realistic diversity found in patients. Mistargeted animals can be identified using behavioral or imaging readouts, or only after being sacrificed leading to smallercohort size after the study has already been concluded. Overall, this method is a rudimentary but effective way to assess a diverse set of PD aspects.

Mesenchymal stem cells modulate misfolded α-synuclein in parkinsonian disorders: A multitarget disease-modifying strategy.

α-Synuclein (α-Syn) aggregates, the major toxic component of Lewy bodies, are proteinaceous fibrillar cytoplasmic inclusions observed in α-synucleinopathies, such as Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Overexpression of α-syn induce neuronal loss and α-syn aggregation in PD animals. Recent studies show that α-syn is released by exocytosis and can be transmitted between brain areas through cell-to-cell propagation. Moreover, aggregates of extracellular α-syn can induce neuroinflammation-mediated neurotoxic signaling through microglial activation and release of pro-inflammatory factors. Thus, modulation of α-syn might be a potential therapeutic strategy for modifying disease progression of α-synucleinopathies. Our previous studies have revealed that MSCs have potent neuroprotective effects in PD animal through modulation of neuroinflammation, inhibition of cell death, and promotion of neurogenesis. Here, we provide further evidence that MSCs have the potential to modulate α-syn-related microenvironments via enhancement of autophagy, proteolysis of α-syn aggregates, inhibition of cell-to-cell transmission of α-syn, stabilization of axonal transport, and phagocytic clearance of α-syn by microglial M2 polarization. With advantages in clinical applications, these data suggests that the use of MSCs as pharmacological modulators of α-syn propagation would be an effective therapeutic approach in PD.

Astrocyte glutamate transporters are increased in an early sporadic model of synucleinopathy

α-Synuclein protein (α-syn) is a central player in Parkinson's disease (PD) and in a spectrum of neurodegenerative diseases collectively known as synucleinopathies. These diseases are characterized by abnormal motor symptoms, such as tremor at rest, slowness of movement, rigidity of posture, and bradykinesia. Histopathological features of PD include preferential loss of dopaminergic neurons in the substantia nigra and formation of fibrillar intraneuronal inclusions called Lewy bodies and Lewy neurites, which are composed primarily of the α-syn protein. Currently, it is well accepted that α-syn oligomers (αSO) are the main toxic agent responsible for the etiology of PD. Glutamatergic excitotoxicity is associated with several neurological disorders, including PD. Excess glutamate in the synaptic cleft can be taken up by the astrocytic glutamate transporters GLAST and GLT-1. Although this event is the main defense against glutamatergic excitotoxicity, the molecular mechanisms that regulate this process have not yet been investigated in an early sporadic model of synucleinopathy. Here, using an early sporadic model of synucleinopathy, we demonstrated that the treatment of astrocytes with αSO increased glutamate uptake. This was associated with higher levels of GLAST and GLT-1 in astrocyte cultures and in a mouse model of synucleinopathy 24 h and 45 days after inoculation with αSO, respectively. Pharmacological inhibition of the TGF-β1 (transforming growth factor beta 1) pathway in vivo reverted GLAST/GLT-1 enhancement induced by αSO injection. Therefore, our study describes a new neuroprotective role of astrocytes in an early sporadic model of synucleinopathy and sheds light on the mechanisms of glutamate transporter regulation for neuroprotection against glutamatergic excitotoxicity in synucleinopathy.

Insights into the development of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from UK estuaries

Hepatocellular fibrillar inclusions (HFI) are an unusual pathology of unknown aetiology affecting European flounder (Platichthys flesus), particularly from estuaries historically impacted by pollution. This study demonstrated that the HFI prevalence range was 6–77% at several UK estuaries, with Spearman rank correlation analysis showing a correlation between HFI prevalence and sediment concentrations of ∑PBDEs and ∑HBCDs. The data showed that males exhibit higher HFI prevalence than females, with severity being more pronounced in estuaries exhibiting higher prevalence. HFI were not age associated indicating a subacute condition. Electron microscopy confirmed that HFI were modified proliferating rough endoplasmic reticulum (RER), whilst immunohistochemistry provided evidence of VTG production in HFI of male P. flesus. Despite positive labelling of aberrant VTG production, we could not provide additional evidence of xenoestrogen exposure. Gene transcripts (VTG/CHR) and plasma VTG concentrations (>1 μg ml−1), were only considered elevated in four male fish showing no correlation with HFI severity. Further analysis revealed that reproductively mature female P. flesus i.e. >3-year-old, did not exhibit HFI, whereas males of all ages were affected. This, combined with previous reports that estradiol (E2) can impair mixed function oxygenase activity, supports a hypothesis that harmful chemical metabolites (following phase 1 metabolism of their parent compounds) are potentially responsible for HFIs observed in male and ≤ 3-year-old female fish. Consequently, HFI and xenoestrogenic induced VTG production could be independent of each other resulting from different concurrent toxicopathic mechanisms, although laboratory exposures will likely be the only way to determine the true aetiology of HFI.

Effect of ethanol as molecular crowding agent on the conformational dynamics of α-synuclein

The functions of many proteins have been directly connected to their conformational changes. The macromolecular crowding environment inside the cell is known to have a significant impact on the equilibria and transition rates between different conformations of the protein. Here we demonstrate the effect of ethanol as crowders on the conformational dynamics of α- synuclein protein, a primary component of the fibrillar neuronal inclusions, and known as Lewy bodies that are diagnostic of Parkinson’s disease. We observed the α-synuclein protein to experience stronger crowding effects with an increase in concentration of ethanol, the crowding agent. The findings that we obtained from this simulation study would serve as valuable guides for expected crowding effects on conformational dynamics of α- synuclein.

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection.

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the “strain” of the conformer that is propagating.

Intranuclear Inclusions in Cerebellar Golgi Cells of Patients with Cerebellar Tumors

Fibrillar Intranuclear inclusions are described in cerebellar Golgi cells of three patients with cerebellar tumors. Cortical biopsies taken during neurosurgical treatment were immediately processed for transmission electron microscopy. The intranuclear inclusion appears as a straight rodlet up to 3 um in length and from 0.4 um in width, immersed in the nucleoplasm and without topographic relationship with the nucleolus. This rodletshows a periodic or crystalloid structure formed by dense bands 9.2 nm thick, separated by clear spaces of 5.4 nm in width (Fig. 2), and in some regions displays a lattice or crystalloid appearance produced by oblique superposition of the dense bands. The findings are discussed in relationship with intranuclear inclusion found in viral and central neurodegeneraive diseases.

Spontaneous Age-Related Histopathological Changes in Microminipigs.

Microminipigs have become an attractive animal model for toxicology and pharmacology studies and for human disease models, owing to their manageable size. Although there are numerous reports of spontaneous age-related lesions in mice, rats, dogs, and monkeys, those in minipigs are scarce. In the present study, spontaneous age-related histopathological changes were investigated using 37 microminipigs (20 males and 17 females) that were 6 months to 10 years of age. Abnormal deposits of materials were evident in several animals from 6 years of age, and these deposits included amyloid in the renal medulla, thyroid gland, and adrenal gland, hyaline droplets in glomeruli, and fibrillar inclusions in neurons. Arterial sclerosing changes (intimal thickening, intimal proliferation, and medial mineralization) and proliferative lesions (hyperplasia of hepatocytes, follicular cells, Leydig cells, and uterine endometrial glands) were present at 4 years of age and beyond. Renal adenoma, uterine leiomyoma, and Leydig cell tumor were observed in several microminipigs. Moreover, glomerulosclerosis, renal interstitial fibrosis, thymic involution, and adrenocortical cell vacuolation were common in aging microminipigs. Since knowledge of age-related changes is helpful for pathologists, the basic information obtained in this study will be a useful reference for all future toxicity evaluations in microminipigs.

Defining α-synuclein species responsible for Parkinson’s disease phenotypes in mice

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated α-synuclein (α-syn). These inclusions are associated with behavioral and pathological PD phenotypes. One strategy for therapeutic interventions is to prevent the formation of these inclusions to halt disease progression. α-Synuclein exists in multiple structural forms, including disordered, nonamyloid oligomers, ordered amyloid oligomers, and fibrils. It is critical to understand which conformers contribute to specific PD phenotypes. Here, we utilized a mouse model to explore the pathological effects of stable β-amyloid-sheet oligomers compared with those of fibrillar α-synuclein. We biophysically characterized these species with transmission EM, atomic-force microscopy, CD spectroscopy, FTIR spectroscopy, analytical ultracentrifugation, and thioflavin T assays. We then injected these different α-synuclein forms into the mouse striatum to determine their ability to induce PD-related phenotypes. We found that β-sheet oligomers produce a small but significant loss of dopamine neurons in the substantia nigra pars compacta (SNc). Injection of small β-sheet fibril fragments, however, produced the most robust phenotypes, including reduction of striatal dopamine terminals, SNc loss of dopamine neurons, and motor-behavior defects. We conclude that although the β-sheet oligomers cause some toxicity, the potent effects of the short fibrillar fragments can be attributed to their ability to recruit monomeric α-synuclein and spread in vivo and hence contribute to the development of PD-like phenotypes. These results suggest that strategies to reduce the formation and propagation of β-sheet fibrillar species could be an important route for therapeutic intervention in PD and related disorders.