From neurodegeneration to neurohomeostasis: the role of ubiquitin

Several years ago ubiquitin immunocytochemistry first demonstrated that ubiquitin protein conjugates are present in intraneuronal inclusions in all the major human chronic neurodegenerative diseases, as well as in inclusions in cerebellar astrocytomas and in hepatocytes in alcoholic liver disease. Unexpectedly, further studies showed that Lewy bodies are present in the cortex. Lewy bodies were originally described in the brain stem and are pathogonomic in the neuropathological diagnosis of Parkinson's disease. A balanced interpretation of further elegant experimental approaches, including transgenesis, suggests that the formation of intraneuronal inclusions is cytoprotective. Putative oligomeric proaggregates (prefibrillar entities) of cellular proteins inhibit the 26S proteasome and promote apoptosis. In the last few years a clutch of distinct experimental approaches have focused on the roles of ubiquitin-related processes in the development of the nervous system and neurohomeostasis. It is now clear that the ubiquitin/proteasome system (UPP) has a pivotal role in synaptogenesis, the formation of neuromuscular junctions and neurotransmitter receptor function. The inhibitory GABA(A) receptor, alpha1 glycine receptor, beta(2)-adrenergic receptor and arrestin, opiate receptors and the excitatory metabotropic glutamate receptor (mGluR1alpha) are regulated by the UPP. It is also increasingly clear that the regulation of long-term synaptic plasticity, and therefore memory, is dependent on both protein synthesis and protein degradation. Therefore, for the first time we have the opportunity to dissect the substrate of memory and the basis of cognitive decline in aging and in chronic neurodegenerative disease. Clearly, further understanding will provide a platform for novel drug development to treat chronic neurodegenerative diseases, including Alzheimer- and Parkinson-related conditions, and possibly psychiatric disorders.     

Classification of protein aggregates

Comparison of protein aggregates/self-associated species between laboratories and across disciplines is complicated by the imprecise language presently used to describe them. In this commentary, we propose a standardized nomenclature and classification scheme that can be applied to describe all protein aggregates. Five categories are described under which a given aggregate may be independently classified: size, reversibility/dissociation, conformation, covalent modification, and morphology. Possible subclassifications within each category, several examples of applications of the nomenclature, and difficulties in making appropriate assignments will be discussed.     

Biophysical characterization of insoluble aggregates of a multi-domain protein: an insight into the role of the various domains

Insoluble (visible) aggregates of a homodimer fusion glycoprotein, consisting of the first extracellular domain of a human protein, fused to the hinge, C(H)2, and C(H)3 domains of a human immunoglobulin G(1) (IgG(1)), were observed during early formulation development. The soluble fraction of the fusion protein was compared to the visible aggregates by various biophysical techniques such as intrinsic and ANS fluorescence emission, reducing and nonreducing SDS-PAGE, equilibrium folding and refolding experiments in urea and guanidine hydrochloride in the absence and presence of mercaptoethanol. Significant differences were observed between the visible aggregates and the supernatant. Partial unfolding of the aggregated molecules was detected by intrinsic and ANS fluorescence. Using urea and guanidine hydrochloride unfolding/solubilization and refolding curves, it was possible to extrapolate that (i) the aggregates were not covalently linked but tightly associated, (ii) the fused domains of the protein were unfolded but not involved in the aggregation process, (iii) the C(H)2 domains were native-like, and (iv) the C(H)3 domains were involved in the aggregation process.     

Effects of protein aggregates: An immunologic perspective

The capacity of protein aggregates to enhance immune responses to the monomeric form of the protein has been known for over a half-century. Despite the clear connection between protein aggregates and antibody mediated adverse events in treatment with early therapeutic protein products such as intravenous immune globulin (IVIG) and human growth hormone, surprisingly little is known about the nature of the aggregate species responsible for such effects. This review focuses on a framework for understanding how aggregate species potentially interact with the immune system to enhance immune responses, garnered from basic immunologic research. Thus, protein antigens presented in a highly arrayed structure, such as might be found in large nondenatured aggregate species, are highly potent in inducing antibody responses even in the absence of T-cell help. Their potency may relate to the ability of multivalent protein species to extensively cross-link B-cell receptor, which (1) activates B cells via Bt kinases to proliferate, and (2) targets protein to class II major histocompatibility complex (MHC)-loading compartments, efficiently eliciting T-cell help for antibody responses. The review further focuses on protein aggregates as they affect an immunogenicity risk assessment, the use of animal models and studies in uncovering effects of protein aggregates, and changes in product manufacture and packaging that may affect generation of protein aggregates.     

The role of citric acid in oral peptide and protein formulations: Relationship between calcium chelation and proteolysis inhibition

The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CA’s calcium chelation activity was developed and verified experimentally using an ion-selective electrode. The effects of CA, its salt (citrate, Cit) and the established permeation enhancer, lauroyl carnitine chloride (LCC) were compared by measuring transepithelial electrical resistance (TEER) and permeability of insulin and FD4 across Caco-2 monolayers and rat small intestinal mucosae mounted in Ussing chambers. Proteolytic degradation of insulin was determined in rat luminal extracts across a range of pH values in the presence of CA. CA’s capacity to chelate calcium decreased ∼10-fold for each pH unit moving from pH 6 to pH 3. CA was an inferior weak permeation enhancer compared to LCC in both in vitro models using physiological buffers. At pH 4.5 however, degradation of insulin in rat luminal extracts was significantly inhibited in the presence of 10 mM CA. The capacity of CA to chelate luminal calcium does not occur significantly at the acidic pH values where it effectively inhibits proteolysis, which is its dominant action in oral peptide formulations. On account of insulin’s low basal permeability, inclusion of alternative permeation enhancers is likely to be necessary to achieve sufficient oral bioavailability since this is a weak property of CA.     

Involvement of unfolded protein responses in neurodegeneration]

Various stresses cause the accumulation of unfolded proteins in the endoplasmic reticulum (ER). To manage the state, cells have the unfolded protein responses (UPR). If the UPR is unsuccessful, ER-mediated apoptosis occurs. To date, three types of UPR, i.e. the induction of chaperones, the translation block, and ER-associated degradation (ERAD) have been reported. To sense the accumulation of unfolded proteins, the ER has IRE1, PERK, and ATF6. The pathways mediated by IRE1 and ATF6 cause the induction of chaperones. The pathway mediated by PERK causes a translation block. The induction of caspase 12, the activation of the JNK pathway, and the induction of CHOP have been reported as apoptosis caused by ER stress. The stability of the cell is based on the balance between UPR and ER-mediated apoptosis. Recently several diseases have been reported to be related to ER stress. We reported that mutant presenilin 1 causes a vulnerability to ER stress because it attenuates the activation of IRE1, PERK, and ATF6. Recent reports have also shown that Parkinson disease and polyglutamine diseases are relevant to ER stress. Therefore it is suggested that the ER stress story is the common mechanism for neurodegerative disorders.     

The neurobiology of sirtuins and their role in neurodegeneration

Sirtuins are highly conserved NAD(+)-dependent enzymes that have beneficial effects against age-related diseases. Aging is the major unifying risk factor for all neurodegenerative disorders. Sirtuins modulate major biological pathways, such as stress response, protein aggregation, and inflammatory processes, that are involved in age-related neurodegenerative diseases. Therefore, sirtuins have been widely studied in the context of the nervous system and neurodegeneration. They are especially interesting because it is possible to alter the activities of sirtuins using small molecules that could be developed into drugs. Indeed, it has been shown that manipulation of SIRT1 activity genetically or pharmacologically impacts neurodegenerative disease models. This review summarizes recent research in sirtuin neurobiology and neurodegenerative diseases and analyzes the potential of therapeutic applications based on sirtuin research.     

The role of iron in neurodegeneration: prospects for pharmacotherapy of Parkinson's disease

Although the aetiology of Parkinson's disease (PD) and related neurodegenerative disorders is still unknown, recent evidence from human and experimental animal models suggests that a misregulation of iron metabolism, iron-induced oxidative stress and free radical formation are major pathogenic factors. These factors trigger a cascade of deleterious events leading to neuronal death and the ensuing biochemical disturbances of clinical relevance. A review of the available data in PD provides the following evidence in support of this hypothesis: (i) an increase of iron in the brain, which in PD selectively involves neuromelanin in substantia nigra (SN) neurons; (ii) decreased availability of glutathione (GSH) and other antioxidant substances; (iii) increase of lipid peroxidation products and reactive oxygen (O2)species (ROS); and (iv) impaired mitochondrial electron transport mechanisms. Most of these changes appear to be closely related to interactions between iron and neuromelanin, which result in accumulation of iron and a continuous production of cytotoxic species leading to neuronal death. Some of these findings have been reproduced in animal models using 6-hydroxydopamine, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), iron loading and beta-carbolines, although none of them is an accurate model for PD in humans. Although it is not clear whether iron accumulation and oxidative stress are the initial events causing cell death or consequences of the disease process, therapeutic efforts aimed at preventing or at least delaying disease progression by reducing the overload of iron and generation of ROS may be beneficial in PD and related neurodegenerative disorders. Current pharmacotherapy of PD, in addition to symptomatic levodopa treatment, includes 'neuroprotective' strategies with dopamine agonists, monoamine oxidase-B inhibitors (MAO-B), glutamate antagonists, catechol O-methyltransferase inhibitors and other antioxidants or free radical scavengers. In the future, these agents could be used in combination with, or partly replaced by, iron chelators and lazaroids that prevent iron-induced generation of deleterious substances. Although experimental and preclinical data suggest the therapeutic potential of these drugs, their clinical applicability will be a major challenge for future research.     

The role of environmental exposures in neurodegeneration and neurodegenerative diseases

Neurodegeneration describes the loss of neuronal structure and function. Numerous neurodegenerative diseases are associated with neurodegeneration. Many are rare and stem from purely genetic causes. However, the prevalence of major neurodegenerative diseases is increasing with improvements in treating major diseases such as cancers and cardiovascular diseases, resulting in an aging population. The neurological consequences of neurodegeneration in patients can have devastating effects on mental and physical functioning. The causes of most cases of prevalent neurodegenerative diseases are unknown. The role of neurotoxicant exposures in neurodegenerative disease has long been suspected, with much effort devoted to identifying causative agents. However, causative factors for a significant number of cases have yet to be identified. In this review, the role of environmental neurotoxicant exposures on neurodegeneration in selected major neurodegenerative diseases is discussed. Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis were chosen because of available data on environmental influences. The special sensitivity the nervous system exhibits to toxicant exposure and unifying mechanisms of neurodegeneration are explored.     

败酱草多糖的脱蛋白研究

目的研究败酱草多糖的脱蛋白工艺。方法采用Sevag法、三氯乙酸法、酶法对败酱草多糖进行脱蛋白研究。结果酶法脱蛋白效果明显好于Sevag法,三氯乙酸法的多糖损失较多。在木瓜蛋白酶用量4%（W/V）、pH值6.0、温度55℃、酶解时间2.5 h的条件下,败酱草多糖的蛋白脱除率为51.65%,多糖损失率为7.93%。结论用酶法脱蛋白效果较好。     

Equilibrium studies of protein aggregates and homogeneous nucleation in protein formulation

Shaking or heat stress may induce protein aggregates. Aggregation behavior of an IgG1 stressed by shaking or heat following static storage at 5 and 25°C was investigated to determine whether protein aggregates exist in equilibrium. Aggregates were detected using different analytical methods including visual inspection, turbidity, light obscuration, size exclusion chromatography, and dynamic light scattering. Significant differences were evident between shaken and heated samples upon storage. Visible and subvisible particles (insoluble aggregates), turbidity and z-average diameter decreased whilst soluble aggregate content increased in shaken samples over time. Insoluble aggregates were considered to be reversible and dissociate into soluble aggregates and both aggregate types existed in equilibrium. Heat-induced aggregates had a denatured protein structure and upon static storage, no significant change in insoluble aggregates content was shown, whilst changes in soluble aggregates content occurred. This suggested that heat-induced insoluble aggregates were irreversible and not in equilibrium with soluble aggregates. Additionally, the aggregation behavior of unstressed IgG1 after spiking with heavily aggregated material (shaken or heat stressed) was studied. The aggregation behavior was not significantly altered, independent of the spiking concentration over time. Thus, neither mechanically stressed native nor temperature-induced denatured aggregates were involved in nucleating or propagating aggregation. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:632–644, 2010     

The crucial role of metal ions in neurodegeneration: the basis for a promising therapeutic strategy

The variety of factors and events involved in neurodegeneration renders the subject a major challenge. Neurodegenerative disorders include a number of different pathological conditions, which share similar critical metabolic processes, such as protein aggregation and oxidative stress, both of which are associated with the involvement of metal ions. In this review, Alzheimer's disease, Parkinson's disease and prion disease are discussed, with the aim of identifying common trends underlying these devastating neurological conditions. Chelation therapy could be a valuable therapeutic approach, since metals are considered to be a pharmacological target for the rationale design of new therapeutic agents directed towards the treatment of neurodegeneration.     

In vitro evidence supporting the therapeutic role of nicotine against neurodegeneration

This review supports the necessity of combining fundamental chemical and biological methods to scrutinize potential causative agents in neurodegeneration. This is supported by recent experimental evidence in relation to the use of nicotine as a potential therapeutic agent, especially when following the path of iron's role in catalysing the generation of reactive oxygen species via a Fenton like reaction. Exploration of the dose-response relationship indicates that acute administration offers the most likely success, reducing tremor and improving cognitive performance amongst others. Confirmation of this relationship is gathered from recent in vivo and in vitro efforts that support this hypothesis.     

Stability and structure of protein-polysaccharide coacervates in the presence of protein aggregates

We have studied at pH 4.2 and three protein (Pr):polysaccharide (Pol) weight ratios (8:1, 2:1 and 1:1) the structure and stability of beta-lactoglobulin/acacia gum/water dispersions containing protein aggregates (BLG/AG/W) or free from aggregates (AF-BLG/AG/W). Phase diagrams were characteristic of complex coacervation. BLG/AG/W dispersions displayed a larger biphasic area than AF-BLG/AG/W dispersions, that moved towards the protein axis. It was concluded that protein aggregates affected complex coacervation both by entropic (size and molecular masses of aggregates) and enthalpic (surface properties of aggregates) effects. Laser light scattering measurements revealed that the particles diameter (d(43)) induced by demixing was controlled by protein aggregates in AF-BLG/AG/W dispersions. At 1 wt.% biopolymer concentration, particles were 15-20 times larger in AF-BLG/AG/W dispersions than in BLG/AG/W dispersions at (Pr:Pol) ratios of 2:1 or 1:1. Confocal scanning laser microscopy showed that AF-BLG/AG/W dispersions only contained spherical coacervates. BLG/AG/W dispersions contained both coacervates and aggregates coated with AG or/and BLG/AG coacervates. At a (Pr:Pol) ratio of 2:1 and 1:1, coacervates were vesicular or multivesicular. Coacervates were smaller in BLG/AG/W dispersions than in AF-BLG/AG/W dispersions. It was concluded that protein aggregates have the intrinsic ability to stabilize complex coacervates and could be used to design multifunctional delivery systems. This study showed that composite dispersions containing both protein aggregates embedded in protein-polysaccharide coacervates and free coacervates may be performed. In this respect, the design of protein aggregates with controlled size distribution and surface properties could open new possibilities both in the non-chemical control of coacervates stability and in the development of multifunctional delivery systems.     

Targeting protein aggregation in neurodegeneration – lessons from polyglutamine disorders

Polyglutamine diseases, such as Huntington’s disease, are among the most common inherited neurodegenerative disorders. They share salient clinical and pathological features with major sporadic neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and amyotropic lateral sclerosis. Over the last decade, protein aggregation has emerged as a common pathological hallmark in neurodegenerative diseases and has, therefore, attracted considerable attention as a likely shared therapeutic target. Because of their clearly defined molecular genetic basis, polyglutamine diseases have allowed researchers to dissect the relationship between neurodegeneration and protein aggregation. In this review, the authors discuss recent progress in understanding polyglutamine-mediated neurotoxicity, and discuss the most promising therapeutic strategies being developed in the polyglutamine diseases and related neurodegenerative disorders.     

A simple method for the detection of insoluble aggregates in protein formulations

Low levels of insoluble aggregates in protein formulations can sometimes only be detected by visual inspection. To overcome the subjectivity and other limitations associated with visual inspection, a microscopic technique based on filtration/staining was developed. This method is a simple modification of the microscopic method listed in USP for particulate matter analysis and it provides two major advantages over the original method. First, particles are easier to see because of the staining. Second, this method is specific to protein aggregates so that it avoids interferences from other nonproteinaceous particles. In addition, this method does not have any restrictions on the rheological or optical properties of the samples. This method can be a useful tool in protein formulation development as demonstrated by its application in the evaluation of monoclonal antibody formulations.     

The role of neuronal nicotinic acetylcholine receptors in acute and chronic neurodegeneration

In the last five years there has been a rapid explosion of publications reporting that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in neurodegenerative disorders. Furthermore, there is a well-established loss of nAChRs in post-mortem brains from patients with Alzheimer's disease, Parkinson's disease and a range of other disorders. In the present review we discuss the evidence that nicotine and subtype selective nAChR ligands can provide neuroprotection in in vitro cell culture systems and in in vivo studies in animal models of such disorders. Whilst in vitro data pertaining to a protective effect of nicotine against nigral neurotoxins like MPTP is less robust, most studies agree that nicotine is protective against glutamate and beta-amyloid toxicity in various culture systems. This effect appears to be mediated by alpha7 subtype nAChRs since the protection is blocked by alpha-bungarotoxin and is mimicked by alpha7 selective agonists. In vivo studies indicate that alpha7 receptors play a critical role in protection from cholinergic lesions and enhancing cognitive function. The exact subtype involved in the neuroprotectant effects seen in animal models of Parkinson's disease is not clear, but in general broad spectrum nAChR agonists appear to provide protection, while alpha4beta2 receptors appear to mediate symptomatic improvements. Evidence favouring a protectant effect of nicotine against acute degenerative conditions is less strong, though some protection has been observed with nicotine pre-treatment in global ischaemia models. A variety of cellular mechanisms ranging from the production of growth factors through to inactivation of toxins and antioxidant actions of nicotine have been proposed to underlie the nAChR-mediated neuroprotection in vitro and in vivo. In summary, although the lack of subtype selective ligands has hampered progress, it is clear that in the future neuronal nAChR agonists could provide functional improvements and slow or halt the progress of several crippling degenerative diseases.