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.     

The role of chaperones in Parkinson's disease and prion diseases

The etiologies of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, polyglutamine diseases, or prion diseases may be diverse; however, aberrations in protein folding, processing, and/or degradation are common features of these entities, implying a role of quality control systems, such as molecular chaperones and the ubiquitin-proteasome pathway. There is substantial evidence for a causal role of protein misfolding in the pathogenic process coming from neuropathology, genetics, animal modeling, and biophysics. The presence of protein aggregates in all neurodegenerative diseases gave rise to the hypothesis that protein aggregates, be it intracellular or extracellular deposits, may perturb the cellular homeostasis and disintegrate neuronal function (Table 1). More recently, however, an increasing number of studies have indicated that protein aggregates are not toxic per se and might even serve a protective role by sequestering misfolded proteins. Specifically, experimental models of polyglutamine diseases, Alzheimer's disease, and Parkinson's disease revealed that the appearance of aggregates can be dissociated from neuronal toxicity, while misfolded monomers or oligomeric intermediates seem to be the toxic species. The unique features of molecular chaperones to assist in the folding of nascent proteins and to prevent stress-induced misfolding was the rationale to exploit their effects in different models of neurodegenerative diseases. This chapter concentrates on two neurodegenerative diseases, Parkinson's disease and prion diseases, with a special focus on protein misfolding and a possible role of molecular chaperones.     

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.     

Cellular pathways leading to neuronal dysfunction and degeneration

There is no cure for devastating neurodegenerative disorders such as Alzheimer's, Parkinson's, Huntington's diseases or amyotrophic lateral sclerosis, which cause longterm suffering and ultimately death. Slowly progressing neurodegenerative diseases affect the lives of many thousands of patients and their families. These disorders are characterized by pathological changes in disease-specific areas of the brain. In each disease, these pathological processes lead to dysfunction and degeneration in distinct subsets of neurons. Research on neurodegenerative disorders has revealed a complex picture of cellular pathology involving abnormalities in biochemical processes, gene regulation, responses to external stimuli, etc. However, despite the differences in the clinical manifestations and selective neuronal vulnerability, on cellular and molecular levels the underlying pathological processes appear similar across different diseases, suggesting common pathways of neurodegeneration. Elucidation of the precise neurodegenerative mechanism(s) is essential for development of effective and safe therapy for these lethal human disorders.     

Removing protein aggregates: the role of proteolysis in neurodegeneration

A common characteristic of neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) is the accumulation of protein aggregates. This reflects a severe disturbance of protein homeostasis, the proteostasis. Here, we review the involvement of the two major proteolytic machineries, the ubiquitin proteasome system (UPS) and the autophagy/lysosomal system, in the pathogenesis of neurodegenerative diseases. These proteolytic systems cooperate to maintain the proteostasis, as is indicated by intricate cross talk. In addition, the UPS and autophagy are regulated by stress pathways that are activated by disturbed proteostasis, like the unfolded protein response (UPR). We will specifically discuss how these proteolytic pathways are affected in neurodegenerative diseases. We will show that there is a differential involvement of the UPS and autophagy in different neurodegenerative disorders. In addition, the proteolytic impairment may be primary or secondary to the pathology. These differences have important implications for the design of therapeutic strategies. The opportunities and caveats of targeting the UPS and autophagy/lysosomal system as a therapeutic strategy in neurodegeneration will be discussed.     

Ubiquitin-proteasome pathway components as therapeutic targets for CNS maladies

In the central nervous system (CNS), abnormal deposition of insoluble protein aggregates or inclusion bodies within nerve cells is commonly observed in association with several neurodegenerative diseases. The ubiquitinated protein aggregates are believed to result from malfunction or overload of the ubiquitin-proteasome pathway or from structural changes in the protein substrates which prevent their recognition and degradation by the ubiquitin-proteasome pathway. Impaired proteolysis might also contribute to the synaptic dysfunction seen early in neurodegenerative diseases because the ubiquitin-proteasome pathway is known to play a role in normal functioning of synapses. Because specificity of the ubiquitin proteasome mediated proteolysis is determined by specific ubiquitin ligases (E3s), identification of specific E3s and their allosteric modulators are likely to provide effective therapeutic targets for the treatment of several CNS disorders. Another unexplored area for the discovery of drug targets is the proteasome. Although many inhibitors of the proteasome are available, no effective drugs exist that can stimulate the proteasome. Since abnormal protein aggregation is a common feature of different neurodegenerative diseases, enhancement of proteasome activity might be an efficient way to remove the aggregates that accumulate in the brain. In this review, we discuss how the components of the ubiquitin-proteasome pathway could be potential targets for therapy of CNS diseases and disorders.     

Rapamycin inhibits polyglutamine aggregation independently of autophagy by reducing protein synthesis

Accumulation of misfolded proteins and protein assemblies is associated with neuronal dysfunction and death in several neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease (HD). It is therefore critical to understand the molecular mechanisms of drugs that act on pathways that modulate misfolding and/or aggregation. It is noteworthy that the mammalian target of rapamycin inhibitor rapamycin or its analogs have been proposed as promising therapeutic compounds clearing toxic protein assemblies in these diseases via activation of autophagy. However, using a cellular model of HD, we found that rapamycin significantly decreased aggregation-prone polyglutamine (polyQ) and expanded huntingtin and its inclusion bodies (IB) in both autophagy-proficient and autophagy-deficient cells (by genetic knockout of the atg5 gene in mouse embryonic fibroblasts). This result suggests that rapamycin modulates the levels of misfolded polyQ proteins via pathways other than autophagy. We show that rapamycin reduces the amount of soluble polyQ protein via a modest inhibition of protein synthesis that in turn significantly reduces the formation of insoluble polyQ protein and IB formation. Hence, a modest reduction in huntingtin synthesis by rapamycin may lead to a substantial decrease in the probability of reaching the critical concentration required for a nucleation event and subsequent toxic polyQ aggregation. Thus, in addition to its beneficial effect proposed previously of reducing polyQ aggregation/toxicity via autophagic pathways, rapamycin may alleviate polyQ disease pathology via its effect on global protein synthesis. This finding may have important therapeutic implications.     

Nanoparticles in the Brain: A Potential Therapeutic System Targeted to an Early Defect Observed in Many Neurodegenerative Diseases

Currently, there are no effective treatments or cures for many neurodegenerative diseases affecting an aging baby-boomer generation. The ongoing problem with many of the current therapeutic treatments is that most are aimed at dissolving or dissociating aggregates and preventing cell death, common neuropathology often seen towards the end stage of disease. Often such treatments have secondary effects that are more devastating than the disease itself. Thus, effective therapeutics must be focused on directly targeting early events such that global deleterious effects of drugs are minimized while beneficial therapeutic effects are maximized. Recent work indicates that in many neurodegenerative diseases long distance axonal transport is perturbed, leading to axonal blockages. Axonal blockages are observed before pathological or behavioral phenotypes are seen indicating that this pathway is perturbed early in disease. Thus, developing novel therapeutic treatments to an early defect is critical in curing disease. Here I review neurodegenerative disease and current treatment strategies, and discuss a novel nanotechnology based approach that is aimed at targeting an early pathway, with the rationale that restoring an early problem will prevent deleterious downstream effects. To accomplish this, knowledge exchange between biologists, chemists, and engineers will be required to manufacture effective novel biomaterials for medical use.     

Targeting mitochondrial dysfunction in neurodegenerative disease: Part I

Importance of the field: The socioeconomic burden of an aging population has accelerated the urgency of novel therapeutic strategies for neurodegenerative disease. One possible approach is to target mitochondrial dysfunction, which has been implicated in the pathogenesis of numerous neurodegenerative disorders.

Areas covered in this review: This review examines the role of mitochondrial defects in aging and neurodegenerative disease, ranging from common diseases such as Alzheimer's and Parkinson's disease to rare familial disorders such as the spinocerebellar ataxias. The review is provided in two parts; in this first part, we discuss the mitochondrial defects that have been most extensively researched: oxidative stress; bioenergetic dysfunction and calcium deregulation.

What the reader will gain: This review provides a comprehensive examination of mitochondrial defects observed in numerous neurodegenerative disorders, discussing therapies that have reached clinical trials and considering potential novel therapeutic strategies to target mitochondrial dysfunction.

Take home message: This is an important area of clinical research, with several novel therapeutics already in clinical trials and many more in preclinical stages. In part II of this review we will focus on possible novel approaches, looking at mitochondrial defects which have more recently been linked to neurodegeneration.     

Protein conformational misfolding and amyloid formation: characteristics of a new class of disorders that include Alzheimer's and Prion diseases

The accumulation of proteinaceous deposits has been recognised to occur in several neurodegenerative conditions including Prion diseases, Alzheimer's disease, Parkinson's disease, and Huntington's disease. Over the last two decades interest in these conditions has increased markedly, fueled partially by an increasing prevalence of these diseases in the Western world. Evidence indicates that anomalous protein misfolding and aggregation, with an accompanying "toxic gain of function" is central to the neuropathogenesis of these diseases. An increased understanding of the similarities and differences in the production, aggregation and accumulation of the respective proteins involved in these diseases, and the associated mechanisms of neurodegeneration, should aid in the development of new therapeutic agents to treat this group of related disorders.     

Optimization of a polyglutamine aggregation inhibitor peptide (QBP1) using a thioflavin T fluorescence assay

Polyglutamine protein aggregates are a hallmark of several neurodegenerative diseases, including Huntington's disease, and increasing evidence suggests that reducing or inhibiting aggregation produces a therapeutic benefit in animal models of disease. Part of the challenge in designing compounds that interfere with protein aggregation is having a sensitive and consistent in vitro assay that allows for efficient screening and lead optimization. Here we describe a simplified polyglutamine assay that uses a soluble, pathological-length polyglutamine construct (62 glutamines [Q62]) fused to glutathione-S-transferase (GST) and measure aggregate formation with fluorescence generated by thioflavin T binding. Controlled release of Q62 from GST using proteolytic cleavage resulted in time-dependent aggregate formation that was not observed for a non-pathological-length GST-Q19 construct. Cleavage of the polyglutamine domain from GST increased the rate of Q62 aggregation from days to hours, significantly decreasing the time for compound analysis. Controlled aggregate formation combined with the fluorescence sensitivity of the dye thioflavin T allowed us to screen a series of peptide analogs for lead optimization of a previously identified peptide aggregation inhibitor, QBP1. QBP1 analogs showed the greatest inhibitory potency when added prior to Q62 aggregate initiation, suggesting that the mechanism of inhibition was interference with early formed aggregates that were not detectable by ultraviolet or dye binding. The assay detected activities that differed by three orders of magnitudes with Z' = 0.56, which is suitable for high-throughput screening and allowed us to do lead optimization of QBP1 analogs for pharmacophore model building.     

TRINUCLEOTIDE-REPEAT EXPANSIONS AND NEURODEGENERATIVE DISEASE: A MECHANISM OF PATHOGENESIS*

1. Studies of a number of hereditary neurodegenerative diseases, the most common of which is Huntington's disease, have identified the expansion of trinucleotide repeats as a common causative mutation. 2. The diseases are caused by expansions of CAG repeats, encoding polyglutamine tracts, within the coding regions of a variety of unrelated genes. The mechanism whereby this specific genetic instability leads to selective neurodegeneration is currently unknown. 3. Our current understanding of these polyglutamine expansion neurodenerative diseases is outlined. A potential mechanism is discussed whereby subtle alterations in glutamine, and consequently glutamate levels, may induce chronic excitotoxicity and slow cell death in neuronal populations possessing specific glutamate receptors. The potential role of glutamate receptor-mediated changes to intracellular calcium levels and energy metabolism in the neurodegenerative pathway is also addressed.     

Asparagine endopeptidase is an innovative therapeutic target for neurodegenerative diseases

Introduction: Asparagine endopeptidase (AEP) is a pH-dependent endolysosomal cysteine protease that cleaves its substrates after asparagine residues. Our most recent study identifies that it possesses the delta-secretase activity, and that it is implicated in numerous neurological diseases such as Alzheimer’s disease (AD) and stroke. Accumulating evidence supports that the inhibition of AEP exhibits beneficial effects for treating these devastating diseases.

Areas covered: Based on recent evidence, it is clear that AEP cleaves its substrate, such as amyloid precursor protein (APP), tau and SET, and plays a critical role in neuronal cell death in various neurodegenerative diseases and stroke. In this article, the basic biology of AEP, its knockout phenotypes in mouse models, its substrates in neurodegenerative diseases, and its small peptidyl inhibitors and prodrugs are discussed. In addition, we discuss the potential of AEP as a novel therapeutic target for neurodegenerative diseases.

Expert opinion: AEP plays a unique role in numerous biological processes, depending on both pH and context. Most striking is our most recent finding; that AEP is activated in an age-dependent manner and simultaneously cleaves both APP and tau, thereby unifying both major pathological events in AD. Thus, AEP acts as an innovative trigger for neurodegenerative diseases. Inhibition of AEP will provide a disease-modifying treatment for neurodegenerative diseases including AD.     

Changing paradigm to target microglia in neurodegenerative diseases: from anti-inflammatory strategy to active immunomodulation

Introduction: The importance of microglia in most neurodegenerative pathologies, from Parkinson’s disease to amyotrophic lateral sclerosis and Alzheimer’s disease, is increasingly recognized. Until few years ago, microglial activation in pathological conditions was considered dangerous to neurons due to its causing inflammation. Today we know that these glial cells also play a crucial physiological and neuroprotective role, which is altered in neurodegenerative conditions.

Areas covered: The neuroinflammatory hypothesis for neurodegenerative diseases has led to the trial of anti-inflammatory agents as therapeutics with largely disappointing results. New information about the physiopathological role of microglia has highlighted the importance of immunomodulation as a potential new therapeutic approach. This review summarizes knowledge on microglia as a potential therapeutic target in the most common neurodegenerative diseases, with focus on compounds directed toward the modulation of microglial immune response through specific molecular pathways.

Expert opinion: Here we support the innovative concept of targeting microglial cells by modulating their activity, rather than simply trying to counteract their inflammatory neurotoxicity, as a potential therapeutic approach for neurodegenerative diseases. The advantage of this therapeutic approach could be to reduce neuroinflammation and toxicity, while at the same time strengthening intrinsic neuroprotective properties of microglia and promoting neuroregeneration.     

Antiapoptotic drugs: a therapautic strategy for the prevention of neurodegenerative diseases

The purpose of this review is to discuss potential pathways involved in the pathogenesis of neurodegenerative diseases, highlighting current pharmacological drug targets in neuronal apoptosis prevention. The incidence of these disorders is expected to rise in the coming years and so finding effective treatments represents a significant challenge for medicine. Alzheimer's disease and Parkinson's disease were both described almost a century ago and are the most important neurodegenerative disorders in the developed world. However, the molecular mechanisms that lead to the development of the neuronal pathology in both diseases are unclear. For this reason, despite substantial research in the area, an effective treatment for these diseases does not yet exist. In the present study we discuss in depth the pathways involved in apoptosis and neuronal death in neurodegenerative diseases. We also examine drugs that may have a neuroprotective effect. Inhibition of apoptosis mediated by oxidative stress generation and mitochondrial alteration or by the blockade of NMDA receptors could constitute a suitable therapeutic strategy for Alzheimer's disease. A multiple therapy with antioxidants, cell cycle inhibitors, GSK3β inhibitors, and STATINS could, in the future, represent a suitable strategy for delaying the progression of neurodegenerative diseases. This research contributes to the development of new methods in the field of apoptosis inhibitors that could provide the future tools for the treatment of Alzheimer's and Parkinson's disease, as well as other neurodegenerative diseases.     

Metabotropic glutamate receptor 5 as a potential therapeutic target in Huntington’s disease

Introduction: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the amino-terminal region of the huntingtin (htt) protein, which underlies the loss of striatal and cortical neurons. Glutamate has been implicated in a number of neurodegenerative diseases, and several studies suggest that the metabotropic glutamate receptor 5 (mGluR5) may represent a target for the treatment of HD.

Areas covered: The main goal of this review is to discuss the current data in the literature regarding the role of mGluR5 in HD and evaluate the potential of mGluR5 as a therapeutic target for the treatment of HD. mGluR5 is highly expressed in the brain regions affected in HD and is involved in movement control. Moreover, mGluR5 interacts with htt and mutated htt profoundly affects mGluR5 signaling. However, mGluR5 stimulation can activate both neuroprotective and neurotoxic signaling pathways, depending on the context of activation.

Expert opinion: Although the data published so far strongly indicate that mGluR5 plays a major role in HD-associated neurodegeneration, htt aggregation and motor symptoms, it is not clear whether mGluR5 stimulation can diminish or intensify neuronal cell loss and HD progression. Thus, future experiments will be necessary to further investigate the outcome of drugs acting on mGluR5 for the treatment of neurodegenerative diseases.     

Targeting aggregation in the development of therapeutics for the treatment of Huntington's disease and other polyglutamine repeat diseases

Huntington's disease (HD) is one of a number of familial polyglutamine (polyQ) repeat diseases. These neurodegenerative disorders are caused by expression of otherwise unrelated proteins that contain an expansion of a polyQ tract, rendering them toxic to specific subsets of vulnerable neurons. These expanded repeats have an inherent propensity to aggregate; insoluble neuronal nuclear and cytoplasmic polyQ aggregates or inclusions are hallmarks of the disorders [1,2]. In HD, inclusions in diseased brains often precede onset of symptoms, and have been proposed to be involved in pathogenicity [3-5]. Various strategies to block the process of aggregation have been developed in an effort to create drugs that decrease neurotoxicity. A discussion of the effect of antibodies, caspase inhibitors, chemical inhibitors, heat-shock proteins, suppressor peptides and transglutaminase inhibitors upon aggregation and disease is presented.