Broad Kinase Inhibition Mitigates Early Neuronal Dysfunction in Tauopathy.

Tauopathies are a group of more than twenty known disorders that involve progressive neurodegeneration, cognitive decline and pathological tau accumulation. Current therapeutic strategies provide only limited, late-stage symptomatic treatment. This is partly due to lack of understanding of the molecular mechanisms linking tau and cellular dysfunction, especially during the early stages of disease progression. In this study, we treated early stage tau transgenic mice with a multi-target kinase inhibitor to identify novel substrates that contribute to cognitive impairment and exhibit therapeutic potential. Drug treatment significantly ameliorated brain atrophy and cognitive function as determined by behavioral testing and a sensitive imaging technique called manganese-enhanced magnetic resonance imaging (MEMRI) with quantitative R1 mapping. Surprisingly, these benefits occurred despite unchanged hyperphosphorylated tau levels. To elucidate the mechanism behind these improved cognitive outcomes, we performed quantitative proteomics to determine the altered protein network during this early stage in tauopathy and compare this model with the human Alzheimer's disease (AD) proteome. We identified a cluster of preserved pathways shared with human tauopathy with striking potential for broad multi-target kinase intervention. We further report high confidence candidate proteins as novel therapeutically relevant targets for the treatment of tauopathy. Proteomics data are available via ProteomeXchange with identifier PXD023562.

Aberrant AZIN2 and polyamine metabolism precipitates tau neuropathology.

Tauopathies display a spectrum of phenotypes from cognitive to affective behavioral impairments; however, mechanisms promoting tau pathology and how tau elicits behavioral impairment remain unclear. We report a unique interaction between polyamine metabolism, behavioral impairment, and tau fate. Polyamines are ubiquitous aliphatic molecules that support neuronal function, axonal integrity, and cognitive processing. Transient increases in polyamine metabolism hallmark the cell's response to various insults, known as the polyamine stress response (PSR). Dysregulation of gene transcripts associated with polyamine metabolism in Alzheimer's disease (AD) brains were observed, and we found that ornithine decarboxylase antizyme inhibitor 2 (AZIN2) increased to the greatest extent. We showed that sustained AZIN2 overexpression elicited a maladaptive PSR in mice with underlying tauopathy (MAPT P301S; PS19). AZIN2 also increased acetylpolyamines, augmented tau deposition, and promoted cognitive and affective behavioral impairments. Higher-order polyamines displaced microtubule-associated tau to facilitate polymerization but also decreased tau seeding and oligomerization. Conversely, acetylpolyamines promoted tau seeding and oligomers. These data suggest that tauopathies launch an altered enzymatic signature that endorses a feed-forward cycle of disease progression. Taken together, the tau-induced PSR affects behavior and disease continuance, but may also position the polyamine pathway as a potential entry point for plausible targets and treatments of tauopathy, including AD.

Spermidine/spermine-N1-acetyltransferase ablation impacts tauopathy-induced polyamine stress response.

BACKGROUND: Tau stabilizes microtubules; however, in Alzheimer's disease (AD) and tauopathies, tau becomes hyperphosphorylated, aggregates, and results in neuronal death. Our group recently uncovered a unique interaction between polyamine metabolism and tau fate. Polyamines exert an array of physiological effects that support neuronal function and cognitive processing. Specific stimuli can elicit a polyamine stress response (PSR), resulting in altered central polyamine homeostasis. Evidence suggests that elevations in polyamines following a short-term stressor are beneficial; however, persistent stress and subsequent PSR activation may lead to maladaptive polyamine dysregulation, which is observed in AD, and may contribute to neuropathology and disease progression. METHODS: Male and female mice harboring tau P301L mutation (rTg4510) were examined for a tau-induced central polyamine stress response (tau-PSR). The direct effect of tau-PSR byproducts on tau fibrillization and oligomerization were measured using a thioflavin T assay and a N2a split superfolder GFP-Tau (N2a-ssGT) cell line, respectively. To therapeutically target the tau-PSR, we bilaterally injected caspase 3-cleaved tau truncated at aspartate 421 (AAV9 Tau ΔD421) into the hippocampus and cortex of spermidine/spermine-N1-acetyltransferase (SSAT), a key regulator of the tau-PSR, knock out (SSAT-/-), and wild type littermates, and the effects on tau neuropathology, polyamine dysregulation, and behavior were measured. Lastly, cellular models were employed to further examine how SSAT repression impacted tau biology. RESULTS: Tau induced a unique tau-PSR signature in rTg4510 mice, notably in the accumulation of acetylated spermidine. In vitro, higher-order polyamines prevented tau fibrillization but acetylated spermidine failed to mimic this effect and even promoted fibrillization and oligomerization. AAV9 Tau ΔD421 also elicited a unique tau-PSR in vivo, and targeted disruption of SSAT prevented the accumulation of acetylated polyamines and impacted several tau phospho-epitopes. Interestingly, SSAT knockout mice presented with altered behavior in the rotarod task, the elevated plus maze, and marble burying task, thus highlighting the impact of polyamine homeostasis within the brain. CONCLUSION: These data represent a novel paradigm linking tau pathology and polyamine dysfunction and that targeting specific arms within the polyamine pathway may serve as new targets to mitigate certain components of the tau phenotype.

Identification of changes in neuronal function as a consequence of aging and tauopathic neurodegeneration using a novel and sensitive magnetic resonance imaging approach.

Tauopathies, the most common of which is Alzheimer's disease (AD), constitute the most crippling neurodegenerative threat to our aging population. Tauopathic patients have significant cognitive decline accompanied by irreversible and severe brain atrophy, and it is thought that neuronal dysfunction begins years before diagnosis. Our current understanding of tauopathies has yielded promising therapeutic interventions but have all failed in clinical trials. This is partly due to the inability to identify and intervene in an effective therapeutic window early in the disease process. A major challenge that contributes to the definition of an early therapeutic window is limited technologies. To address these challenges, we modified and adapted a manganese-enhanced magnetic resonance imaging (MEMRI) approach to provide sensitive and quantitative power to detect changes in broad neuronal function in aging mice. Considering that tau tangle burden correlates well with cognitive impairment in Alzheimer's patients, we performed our MEMRI approach in a time course of aging mice and an accelerated mouse model of tauopathy. We measured significant changes in broad neuronal function as a consequence of age, and in transgenic mice, before the deposition of bona fide tangles. This MEMRI approach represents the first diagnostic measure of neuronal dysfunction in mice. Successful translation of this technology in the clinic could serve as a sensitive diagnostic tool for the definition of effective therapeutic windows.

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative-associated proteins.

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans-synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease-associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP-43, α-synuclein, and the microtubule-associated protein tau, can be driven out of the cell by an Hsc70 co-chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.

Neurodegeneration and the Heat Shock Protein 70 Machinery: Implications for Therapeutic Development.

The growing prevalence of individuals diagnosed with neurodegenerative disorders has brought into sharp relief the lack of treatment options for individuals struggling with these diseases. As more is discovered about the mechanisms of these neurodegenerative conditions, increasing evidence indicates a common theme in these proteinopathies is altered cellular protein homeostasis. In particular, the interactions of disease-associated proteins with the major cellular chaperones, heat shock proteins90 kDa and 70 kDa (Hsp90/Hsp70), are changed. Therefore, a promising strategy for therapeutic intervention is chemical inhibition and modification of these molecular chaperone proteins. Here we review the rationale behind therapeutic strategies targeting Hsp70 and its complement of co-chaperones, and describe the current literature regarding the use of small molecule inhibitors of Hsp70 in models of neurodegenerative disease.

Noncontact Rotational Head Injury Produces Transient Cognitive Deficits but Lasting Neuropathological Changes.

Traumatic brain injury (TBI) caused by improvised explosive devices (IEDs) is a growing problem in military settings, but modeling this disease in rodents to pre-clinically evaluate potential therapeutics has been challenging because of inconsistency between models. Although the effects of primary blast wave injury have been extensively studied, little is known regarding the effects of noncontact rotational TBIs independent of the blast wave. To model this type of injury, we generated an air cannon system that does not produce a blast wave, but generates enough air pressure to cause rotational TBI. Mice exposed to this type of injury showed deficits in cognitive and motor task acquisition within 1-2 weeks post-injury, but mice tested 7-8 weeks post-injury did not retain any deficits. This suggests that the effects of a single, noncontact rotational TBI are not long lasting. Despite the transient nature of the behavioral deficits, increased levels of phosphorylated tau were observed at 2 and 8 weeks post-injury; however, this tau did not adopt typical pathological structures that have been observed in other TBI models that incorporate blast waves. This was possibly attributed to the fact that this injury was insufficient to induce changes in microglial activation, which was not affected at 2 or 8 weeks post-injury. Taken together, these data suggest that exposure to noncontact, rotational head injury only produces transient cognitive anomalies, but elicits some minor lasting neuropathological changes.

Pathological Tau Promotes Neuronal Damage by Impairing Ribosomal Function and Decreasing Protein Synthesis.

One of the most common symptoms of Alzheimer's disease (AD) and related tauopathies is memory loss. The exact mechanisms leading to memory loss in tauopathies are not yet known; however, decreased translation due to ribosomal dysfunction has been implicated as a part of this process. Here we use a proteomics approach that incorporates subcellular fractionation and coimmunoprecipitation of tau from human AD and non-demented control brains to identify novel interactions between tau and the endoplasmic reticulum (ER). We show that ribosomes associate more closely with tau in AD than with tau in control brains, and that this abnormal association leads to a decrease in RNA translation. The aberrant tau-ribosome association also impaired synthesis of the synaptic protein PSD-95, suggesting that this phenomenon contributes to synaptic dysfunction. These findings provide novel information about tau-protein interactions in human brains, and they describe, for the first time, a dysfunctional consequence of tau-ribosome associations that directly alters protein synthesis. Significance statement: Despite the identification of abnormal tau-ribosomal interactions in tauopathies >25 years ago, the consequences of this association remained elusive until now. Here, we show that pathological tau associates closely with ribosomes in AD brains, and that this interaction impairs protein synthesis. The overall result is a stark reduction of nascent proteins, including those that participate in synaptic plasticity, which is crucial for learning and memory. These data mechanistically link a common pathologic sign, such as the appearance of pathological tau inside brain cells, with cognitive impairments evident in virtually all tauopathies.

Targeting the ER-autophagy system in the trabecular meshwork to treat glaucoma.

A major drainage network involved in aqueous humor dynamics is the conventional outflow pathway, which is gated by the trabecular meshwork (TM). The TM acts as a molecular sieve, providing resistance to aqueous outflow, which is responsible for regulating intraocular pressure (IOP). If the TM is damaged, aqueous outflow is impaired, IOP increases and glaucoma can manifest. Mutations in the MYOC gene cause hereditary primary open-angle glaucoma (POAG) by promoting the abnormal amyloidosis of the myocilin protein in the endoplasmic reticulum (ER), leading to ER stress-induced TM cell death. Myocilin accumulation is observed in approximately 70-80% of all glaucoma cases suggesting that environmental or other genetic factors may also promote myocilin toxicity. For example, simply preventing myocilin glycosylation is sufficient to promote its abnormal accretion. These myocilin amyloids are unique as there are no other known pathogenic proteins that accumulate within the ER of TM cells and cause toxicity. Moreover, this pathogenic accumulation only kills TM cells, despite expression of this protein in other cell types, suggesting that another modifier exclusive to the TM participates in the proteotoxicity of myocilin. ER autophagy (reticulophagy) is one of the pathways essential for myocilin clearance that can be impacted dramatically by aging and other environmental factors such as nutrition. This review will discuss the link between myocilin and autophagy, evaluating the role of this degradation pathway in glaucoma as well as its potential as a therapeutic target.

The active Hsc70/tau complex can be exploited to enhance tau turnover without damaging microtubule dynamics.

The pathological accumulation of abnormally hyperphosphorylated and aggregated tau, a neuronal microtubule (MT)-associated protein that functions to maintain MT stability, is implicated in a number of hereditary and sporadic neurodegenerative diseases including frontotemporal dementia and Alzheimer's disease. Targeting tau for the treatment of these diseases is an area of intense interest and toward that end, modulation of cellular molecular chaperones is a potential therapeutic target. In particular, the constitutive Hsp70 isoform, Hsc70, seems highly interconnected with tau, preserving tau protein levels and synergizing with it to assemble MTs. But the relationship between tau and Hsc70, as well as the impact of this interaction in neurons and its therapeutic implications remain unknown. Using a human dominant negative Hsc70 that resembles isoform selective inhibition of this important chaperone, we found for the first time that Hsc70 activity is required to stimulate MT assembly in cells and brain. However, surprisingly, active Hsc70 also requires active tau to regulate MT assembly in vivo, suggesting that tau acts in some ways as a co-chaperone for Hsc70 to coordinate MT assembly. This was despite tau binding to Hsc70 as substrate, as determined biochemically. Moreover, we show that while chronic Hsc70 inhibition damaged MT dynamics, intermittent treatment with a small molecule Hsp70 inhibitor lowered tau in brain tissue without disrupting MT integrity. Thus, in tauopathies, where MT injury would be detrimental to neurons, the unique relationship of tau with the Hsc70 machinery can be exploited to deplete tau levels without damaging MT networks.

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement.

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.

Cellular factors modulating the mechanism of tau protein aggregation.

Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer's disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer's disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones.

Synthesis, stereochemical analysis, and derivatization of myricanol provide new probes that promote autophagic tau clearance.

We previously discovered that one specific scalemic preparation of myricanol (1), a constituent of Myrica cerifera (bayberry/southern wax myrtle) root bark, could lower the levels of the microtubule-associated protein tau (MAPT). The significance is that tau accumulates in a number of neurodegenerative diseases, the most common being Alzheimer's disease (AD). Herein, a new synthetic route to prepare myricanol using a suitable boronic acid pinacol ester intermediate is reported. An X-ray crystal structure of the isolated myricanol (1) was obtained and showed a co-crystal consisting of (+)-aR,11S-myricanol (2) and (-)-aS,11R-myricanol (3) coformers. Surprisingly, 3, obtained from chiral separation from 1, reduced tau levels in both cultured cells and ex vivo brain slices from a mouse model of tauopathy at reasonable mid-to-low micromolar potency, whereas 2 did not. SILAC proteomics and cell assays revealed that 3 promoted tau degradation through an autophagic mechanism, which was in contrast to that of other tau-lowering compounds previously identified by our group. During the course of structure-activity relationship (SAR) development, we prepared compound 13 by acid-catalyzed dehydration of 1. 13 had undergone an unexpected structural rearrangement through the isomyricanol substitution pattern (e.g., 16), as verified by X-ray structural analysis. Compound 13 displayed robust tau-lowering activity, and, importantly, its enantiomers reduced tau levels similarly. Therefore, the semisynthetic analogue 13 provides a foundation for further development as a tau-lowering agent without its SAR being based on chirality.

Age-associated epigenetic upregulation of the FKBP5 gene selectively impairs stress resiliency.

Single nucleotide polymorphisms (SNPs) in the FK506 binding protein 5 (FKBP5) gene combine with traumatic events to increase risk for post-traumatic stress and major depressive disorders (PTSD and MDD). These SNPs increase FKBP51 protein expression through a mechanism involving demethylation of the gene and altered glucocorticoid signaling. Aged animals also display elevated FKBP51 levels, which contribute to impaired resiliency to depressive-like behaviors through impaired glucocorticoid signaling, a phenotype that is abrogated in FKBP5-/- mice. But the age of onset and progressive stability of these phenotypes remain unknown. Moreover, it is unclear how FKBP5 deletion affects other glucocorticoid-dependent processes or if age-associated increases in FKBP51 expression are mediated through a similar epigenetic process caused by SNPs in the FKBP5 gene. Here, we show that FKBP51-mediated impairment in stress resiliency and glucocorticoid signaling occurs by 10 months of age and this increased over their lifespan. Surprisingly, despite these progressive changes in glucocorticoid responsiveness, FKBP5-/- mice displayed normal longevity, glucose tolerance, blood composition and cytokine profiles across lifespan, phenotypes normally associated with glucocorticoid signaling. We also found that methylation of Fkbp5 decreased with age in mice, a process that likely explains the age-associated increases in FKBP51 levels. Thus, epigenetic upregulation of FKBP51 with age can selectively impair psychological stress-resiliency, but does not affect other glucocorticoid-mediated physiological processes. This makes FKBP51 a unique and attractive therapeutic target to treat PTSD and MDD. In addition, aged wild-type mice may be a useful model for investigating the mechanisms of FKBP5 SNPs associated with these disorders.

Exploiting the interaction between Grp94 and aggregated myocilin to treat glaucoma.

Gain-of-function mutations in the olfactomedin domain of the MYOC gene facilitate the toxic accumulation of amyloid-containing myocilin aggregates, hastening the onset of the prevalent ocular disorder primary open-angle glaucoma. Aggregation of wild-type myocilin has been reported in other glaucoma subtypes, suggesting broader relevance of misfolded myocilin across the disease spectrum, but the absence of myocilin does not cause disease. Thus, strategies aimed at eliminating myocilin could be therapeutically relevant for glaucoma. Here, a novel and selective Grp94 inhibitor reduced the levels of several mutant myocilin proteins as well as wild-type myocilin when forced to misfold in cells. This inhibitor rescued mutant myocilin toxicity in primary human trabecular meshwork cells. Mechanistically, in vitro kinetics studies demonstrate that Grp94 recognizes on-pathway aggregates of the myocilin olfactomedin domain (myoc-OLF), accelerates rates of aggregation and co-precipitates with myoc-OLF. These results indicate that aberrant myocilin quaternary structure drives Grp94 recognition, rather than peptide motifs exposed by unfolded protein. Inhibition of Grp94 ameliorates the effects of Grp94-accelerated myoc-OLF aggregation, and Grp94 remains in solution. In cells, when wild-type myocilin is driven to misfold and aggregate, it becomes a client of Grp94 and sensitive to Grp94 inhibition. Taken together, the interaction of Grp94 with myocilin aggregates can be manipulated by cellular environment and genetics; this process can be exploited with Grp94 inhibitors to promote the clearance of toxic forms of myocilin.

Accelerated neurodegeneration through chaperone-mediated oligomerization of tau.

Aggregation of tau protein in the brain is associated with a class of neurodegenerative diseases known as tauopathies. FK506 binding protein 51 kDa (FKBP51, encoded by FKBP5) forms a mature chaperone complex with Hsp90 that prevents tau degradation. In this study, we have shown that tau levels are reduced throughout the brains of Fkbp5-/- mice. Recombinant FKBP51 and Hsp90 synergized to block tau clearance through the proteasome, resulting in tau oligomerization. Overexpression of FKBP51 in a tau transgenic mouse model revealed that FKBP51 preserved the species of tau that have been linked to Alzheimer's disease (AD) pathogenesis, blocked amyloid formation, and decreased tangle load in the brain. Alterations in tau turnover and aggregate structure corresponded with enhanced neurotoxicity in mice. In human brains, FKBP51 levels increased relative to age and AD, corresponding with demethylation of the regulatory regions in the FKBP5 gene. We also found that higher FKBP51 levels were associated with AD progression. Our data support a model in which age-associated increases in FKBP51 levels and its interaction with Hsp90 promote neurotoxic tau accumulation. Strategies aimed at attenuating FKBP51 levels or its interaction with Hsp90 have the potential to be therapeutically relevant for AD and other tauopathies.

Nuclear localization of clathrin involves a labile helix outside the trimerization domain.

Clathrin is a trimeric protein involved in receptor-mediated-endocytosis, but can function as a non-trimer outside of endocytosis. We have discovered that the subcellular distribution of a clathrin cysteine mutant we previously studied is altered and a proportion is also localized to nuclear spaces. MALS shows C1573A hub is a mixture of trimer-like and detrimerized molecules. The X-ray structure of the trimerization domain reveals that without light chains, a helix harboring cysteine-1573 is reoriented. We propose clathrin has a detrimerization switch, which suggests clathrin topology can be altered naturally for new functions.

Replacement of charged and polar residues in the coiled-coiled interface of huntingtin-interacting protein 1 (HIP1) causes aggregation and cell death.

HIP1 crystal structures solved in our laboratory revealed abnormalities in the coiled-coil region, suggesting intrinsic plasticity. To test this, specific amino acids in the coiled-coil were mutated. The apparent thermal stability of HIP1 was altered when Thr528 and Glu531 were replaced by leucine, and was enhanced when Lys510 was also mutated. In cells, HIP1 mutant expression produced aggregation. MTS and flow cytometry indicate a correlation between aggregated HIP1 and enhanced cell death. These data support the idea that flexibility of the HIP1 coiled-coil domain is important for normal function and may lead to new insights into Huntington's disease.

The effects of prion protein expression on metal metabolism.

The prion protein is a glycoprotein that binds metals such as copper and manganese. When converted to a proteinase resistant isoform it is associated with prion diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Although, the co-ordination and metal affinity of the prion protein has been well studied, the association of the protein with cellular metal metabolism has been less well investigated. We used transgenic manipulation of prion protein expression and other recombinant techniques to alter expression of known copper binding proteins to investigate the role of the prion protein in copper metabolism. We found that changing the expression of the prion protein alters proteins associated with copper uptake, storage and export from the cell. In addition, alteration in the expression of superoxide dismutases increased prion protein expression dramatically. Reducing copper in the diet decreased expression of the prion protein in the brain while increased dietary manganese dramatically increased the protein's expression. Cellular prion infection also increased the expression of metal transporting proteins and increased cellular manganese concentrations. Overall our results show a close link between cellular resistance to oxidative stress and also copper metabolism. These findings are in line with previous data suggesting that the prion protein is an antioxidant and associated with copper uptake into cells. The disturbance to copper metabolism, as a result of altered prion protein expression clearly demonstrates the important role of the prion protein in copper metabolism. The implication is that prion protein expression has a homeostatic role in copper metabolism.

Mechanisms of prion protein aggregation.

The prion protein is a cell surface glycoprotein that is converted to a protease resistant abnormal isoform during the course of prion disease. The normal isoform of this protein has been shown to be an antioxidant that aids the survival of neurones. The abnormal isoform is associated with both the transmissible agent of prion diseases and is also toxic. Recent studies have shown that there are multiple end states in terms of aggregation of the protein. Both soluble oligomers and insoluble fibrils can form from the abnormal isoform. Although fibrils are characteristic of the disease, the most infectious prions are associated with oligomers. Neurotoxicity can be associated with fibrils but mostly appears to be due to small aggregates. For many years fibrils were believed to be central to the disease process but currently evidence supports the notion that fibrils represent a "bulk" form of abnormal protein, which is largely inert, but carried along a small active component. This review will examine what is known about the mechanisms behind prion protein aggregation, and the relevance of each form for the disease.