Lynn Bedford

David Hay

Simon Paine

Nooshin Rezvani

Maureen Mee

James Lowe

John Mayer
email: john.mayer@nottingham.ac.uk

Is

Keywords: ; ubiquitin, 26S proteasomes, neurodegeneration, dementia with Lewy bodies, Parkinson's disease

Introduction

Walk in the street of any city in the world and you will notice more and more ageing people. Demographic predictions indicate that the ageing population is set to grow further and further, with centenarians becoming commonplace in many countries of the world [START_REF] Kincannon | Demography of aging in China and the United States and the economic well-being of their older populations[END_REF]. The increasing numbers of the ageing population can be attributed to the control of childhood and adult infectious disease, public health improvements and good nutrition. This creates new problems for society, ranging from paying pensions for ever increasing periods to the construction of residential or nursing homes for the elderly. The only robust predictable correlation of dementing illness is age.

The brain has been described as a multi-organ system.

Irrespective of this overarching notion, the brain shows profound structural and functional complexity as might be expected to control the behavioral and physiological activities of the body. There are several neurotransmitters that via different neuronal receptors regulate electrical activity in different parts of the brain. The neurotransmitters include glutamate, acetylcholine, serotonin, γ-amino butyric acid and dopamine. Deficits in dopamine were detected many years ago in regions of the brain associated with Parkinson's disease [START_REF] Hornykiewicz | Dopamine (3-hydroxytyramine) in the central nervous system and its relation to the Parkinson syndrome in man[END_REF][START_REF] Hornykiewicz | The discovery of dopamine deficiency in the parkinsonian brain[END_REF]. This lead to the use of levo-dopa and dopa decarboxylase inhibitors to treat Parkinson's disease. The use of these drugs and others to treat Parkinson's disease

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does not prevent neuronal death: disease progression is irreversible. The findings in Parkinson's disease led to the search for neurochemical deficits in the brain in other neurodegenerative diseases. Declining acetylcholine was found in certain parts of the brain in Alzheimer-related disorders, including Alzheimer's disease and dementia with Lewy bodies [START_REF] Mann | Is the loss of cerebral cortical choline acetyl transferase activity in Alzheimer's disease due to degeneration of ascending cholinergic nerve cells[END_REF]. In turn, these observations led to the development of acetyl cholinesterase inhibitors to treat the symptoms of these diseases.

In order to try and gain an overall understanding of the molecular changes in chronic neurodegenerative disease it is necessary to remember that in the ageing brain there are a variety of neuropathological problems that are associated with cognitive decline and movement disorders, in addition to the neuronal death. These pathologies may appear as hallmarks that characterise a specific disease, e.g. proteinaceous aggregates such as Lewy bodies in neurons in the substania nigra in Parkinson's disease or neurofibrillary tangles in neurons in Alzheimer's disease. However, it is increasingly clear that these pathologies can be present together in the brains of many patients presenting with cognitive decline [START_REF] Burns | Clinical practice with anti-dementia drugs: consensus statement from British Association for Pyschopharamacology[END_REF]. If drugs are to be developed to target fundamental defects in cellular physiology, it will be important to Emphasis must be placed on the key, sometimes overlooked, major pathological finding in the ageing brain, neuronal death. This predominant event in the brain in Alzheimer's disease and Parkinson's disease has so-far not been recapitulated in transgenic models of disease. Animal models have been generated to over-express normal or mutated versions of the proteins that accumulate in aggregates in the diseases without extensive neurodegeneration [START_REF] Mcgowan | A decade of modeling Alzheimer's disease in transgenic mice[END_REF]. There is much in vitro experimental evidence that fragments of the Alzheimer precursor protein can cause cell death in neuronal cell lines or primary neurons [START_REF] Shearman | Toxicity of protein aggregates in PC12 cells: 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay[END_REF], but this does not generally occur in transgenic animals expressing large quantities of these so-called amyloidogenic proteins. Real understanding of chronic neurodegeneration will only come about when there is experimental insight into the mechanisms of neuronal cell death together with intraneuronal inclusion body and extraneuronal amyloid formation.

The Lewy bodies that are found in surviving neurons in the substantia nigra in Parkinson's disease and cortex in dementia with Lewy bodies contain α-synuclein and ubiquitylated proteins. Putative

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precursors of Lewy bodies (pale bodies) also contain mitochondria. The diseases are characterized by extensive neuronal cell death. How can this be explained? Furthermore for progress in understanding and treating disease can these neuropathological features be recapitulated in animal models?

The ubiquitin proteasome system

Protein ubiquitylation/deubiquitylation has moved from the periphery to centre stage in cell physiology, cooperating and competing with protein phosphorylation/dephosphorylation in the control of cellular activities. Ubiquitin was initially intensely studied as a tag for the degradation of target proteins by the 26S proteasome [START_REF] Glickman | The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction[END_REF]. Regulated protein degradation is a major force in the cell to control the concentration of key regulatory proteins like transcription factors.

Abnormal degradation of such molecules plays an important role in cancer [START_REF] Devoy | The ubiquitin-proteasome system and cancer Essays[END_REF]. However, protein ubiquitylation has many other roles in eukaryotic cells [START_REF] Welchman | Ubiquitin and ubiquitin-like proteins as multifunctional signals[END_REF]. Proteins are monophosphorylated on serines, threonines and tyrosines to achieve regulatory diversity in the cell.

Proteins can be monoubiquitylated on single lysine residues, polyubiquitylated on single or multiple lysines, e.g. p53 [START_REF] Brooks | p53 ubiquitination: Mdm2 and beyond[END_REF] or multiubiquitylated on different lysines to achieve regulatory diversity in the cell [12].

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The 26S proteasome consists of a cylindrical multi-subunit 20S catalytic core (α 7 β 7 β 7 α 7 ) together with two 19S end caps. Each 19S particle consists of a nine subunit base and a lid containing multiple nonidentical subunits, involved in functions including the recognition of polyubiquitylated proteins destined for degradation [START_REF] Goldberg | The cellular chamber of doom[END_REF]. The base contains six related, but non-identical and non-redundant ATPases of the AAA superfamily of ATPases (ATPases of Alternative Activities). The putative heterohexameric ring of ATPases is a protein unfolding machine to feed string-like polypeptides into the catalytic core of the 26S proteasome [START_REF] Braun | The base of the proteasome regulatory particle exhibits chaperonelike activity[END_REF] after deubiquitylation by proteasomal deubiquitylating enzymes. Two of the non-ATPases in the base appear to form a central "stub" composed of rpn2 attached to the 20S proteolytic core with rpn1 sitting on top of rpn2 [START_REF] Rosenzweig | The central unit within the 19S regulatory particle of the proteasome[END_REF]. The rpn2 protein interacts with the Nterminus of rpn13, which is a receptor for ubiquitylated proteins [START_REF] Husnjak | Proteasome subunit Rpn13 is a novel ubiquitin receptor[END_REF].

The C-terminus of rpn13 is attached to deubiquitylating enzyme Uch37.

It is tempting to speculate that the "stub" acts as a central hub separated by an annulus from the putative hexameric ATPases. Target proteins could be unfolded in the central annulus between the ATPase ring and the central "stub" following deubiquitylation to produce the string-like polypeptides for insertion into the 20S catalytic core. Such mechanical coupling of ATPase activity to protein movement is

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reminiscent of the rotary biochemical mechanism of the mitochondrial F1-ATPase [START_REF] Berg | Keeping up with the F1-ATPase[END_REF].

Fig. 1 (the 26S proteasome)

Other forms of proteasomes also exist in the cell [START_REF] Knowlton | Structure of the proteasome activator REGalpha (PA28alpha)[END_REF] and it is increasingly seen that proteasomes have many roles. For example, in the regulation of transcription where 26S proteasomes, 20S proteasomes, 19S particles and sub-particles, ATPases independent of 20S (APIS) [START_REF] Gonzalez | Recruitment of a 19S proteasome subcomplex to an activated promoter[END_REF] are associated with transcriptional complexes for different genes [START_REF] Muratani | How the ubiquitin-proteasome system controls transcription[END_REF][START_REF] Auld | Genomic association of the proteasome demonstrates overlapping gene regulatory activity with transcription factor substrates[END_REF][START_REF] Sikder | Widespread, but non-identical, association of proteasomal 19 and 20 S proteins with yeast chromatin[END_REF].

The ubiquitin proteasome system (UPS) has many important roles in neuronal development and homeostasis [START_REF] Bingol | Synaptic protein degradation by the ubiquitin proteasome system[END_REF][START_REF] Bingol | Activity-dependent dynamics and sequestration of proteasomes in dendritic spines[END_REF]. Synaptogenesis and synaptic plasticity are, in part, regulated by the UPS [START_REF] Ehlers | Eppendorf 2003 prize-winning essay. Ubiquitin and the deconstruction of synapses[END_REF]. Synaptic functions and electrical activity are regulated by the combined processes of protein synthesis and protein degradation by the UPS [START_REF] Bingol | Synaptic protein degradation by the ubiquitin proteasome system[END_REF][START_REF] Jiang | Regulation and function of local protein sysnthesis in neuronal dendrites[END_REF]. In turn, normal behaviors and memory [START_REF] Lee | Synaptic protein degradation underlies destabilization of retrieved fear memory[END_REF] are regulated by the UPS. The functions and stability of neurotransmitter receptors and the organization of the post-synaptic density are controlled by the UPS [START_REF] Ehlers | Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system[END_REF]. Protein synthesis and degradation are not confined to the neuronal cell body but also occur in synapses to control synaptic morphology and electrical activity.

Protein degradation in all cells is not mediated by one mechanism.

In addition to the UPS, which degrades cytosolic and nuclear proteins,

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there is the autophagic and endosome-lysosome system. Interestingly, autophagy is controlled by an enzyme cascade involving ubiquitons, cousins of ubiquitin [START_REF] Welchman | Ubiquitin and ubiquitin-like proteins as multifunctional signals[END_REF]. The endosome-lysosome system is likewise regulated by ubiquitin, through the ubiquitylation of the tails of ligandbound receptors to ensure internalization along with other posttranslation modifications, e.g. phosphorylation [START_REF] Welchman | Ubiquitin and ubiquitin-like proteins as multifunctional signals[END_REF][START_REF] Haglund | Multiple monoubiquitylation of RTKs is sufficient for their endocytosis and degradation[END_REF][START_REF] Polo | A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins[END_REF]. Protein ubiquitylation is also needed for the delivery of cargo into multivesicular bodies -via the so-called ESCRT complexes (endosomal sorting complexes required for transport) for fusion with lysosomes and destruction of the contents of the multivesicular bodies [START_REF] Katzmann | Receptor downregulation and multivesicular-body sorting[END_REF]. It is interesting and perhaps evolutionarily predictable, that ubiquitons should have been used to control all the known protein degradation systems in the cell (except perhaps the mitochondrial degradation systems?).

The UPS and other protein degradation systems may have acquired particular importance in the nervous system because, in general, adult neurons do not divide and therefore protein elimination from neurons assumes particular importance in situations where proteins become damaged, e.g. oxidized or undergo structural changes such as conformational changes as in chronic neurodegenerative disease.

The first indications of the activity of the UPS in neurons in disease was the detection by ubiquitin immunohistochemistry of ubiquitylated proteins in protein inclusions in all the major chronic

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neurodegenerative diseases [reviewed in [START_REF] Lowe | Ubiquitin in neurodegenerative diseases[END_REF]]. Furthermore, the detection of cortical Lewy bodies by ubiquitin immunocytochemistry facilitated the identification and further clinical definition of dementia with Lewy bodies as a chronic neurodegenerative disease [START_REF] Lennox | Diffuse Lewy body disease: an important differential diagnosis in dementia with extrapyramidal features[END_REF][START_REF] Lennox | Anti-ubiquitin immunocytochemistry is more sensitive than conventional techniques in the detection of diffuse Lewy body disease[END_REF][START_REF] Lowe | Ubiquitin is a common factor in intermediate filament inclusion bodies of diverse type in man, including those of Parkinson's disease, Pick's disease, and Alzheimer's disease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and mallory bodies in alcoholic liver disease[END_REF][START_REF] Mayer | Ubiquitin and dementia[END_REF]. As is usual in biomedical science, the difficulty has been to explain why ubiquitylated proteins accumulate in protein aggregates in all the different chronic neurodegenerative diseases. There could be several explanations for accumulation of ubiquitylated proteins in inclusions, including malfunction or overwhelming of the 26S proteasome or ubiquitylation of proteins as part of a signaling pathway for cell survival or death. Ubiquitylated proteins may accumulate in inclusions as part of a mechanism, possibly related to the "aggresome" response, to protect neurons from cell death. Alternatively, as some believe, protein ubiquitylation may be a late event in inclusion biogenesis and perhaps an epiphenomenon in relation to disease progression and cell death. roles, e.g. in the heart [START_REF] Wang | Regulation of cellular functions by the ERK5 signalling pathway[END_REF], thymocytes [START_REF] Sohn | Non-redundant function of the MEK5-ERK5 pathway in thymocyte apoptosis[END_REF] and during angiogenesis [START_REF] Pi | BMK1/ERK5 is a novel regulator of angiogenesis by destabilizing hypoxia inducible factor 1alpha[END_REF], but also functions in neuronal survival mechanisms in response to retrogradely transported nerve growth factor [START_REF] Watson | Neurotrophins use the Erk5 pathway to mediate a retrograde survival response[END_REF] and in response to neuronal oxidative damage [START_REF] Wang | Regulation of cellular functions by the ERK5 signalling pathway[END_REF]. Immunohistochemistry with antibodies to MEK5 and ERK5 showed that both neurofibrillary tangles and Lewy bodies contain MEK5 and the afflicted perikarya are enriched in ERK5 [START_REF] Mayer | Methods and compositions relating to diagnosis and treatment of progressive neurodegenerative disease UK Patent application 0606432[END_REF]. These findings provide evidence for a potential generic survival pathway in response to protein aggregates, which appears to take place in neurons containing the inclusions associated with chronic neurodegenerative disease. Neurofibrillary tangles and Lewy bodies contain the p62 protein, which is also involved in signaling in the cell, e.g. in the NFΚB system [START_REF] Kuusisto | Ubiquitin-binding protein p62 expression is induced during apoptosis and proteasomal inhibition in neuronal cells[END_REF][START_REF] Ciani | Structure of the ubiquitin-associated domain of p62 (SQSTM1) and implications for mutations that cause Paget's disease of bone[END_REF]. A direct molecular connection between the presence of ubiquitylated proteins in inclusions and MEK5 can be made through the p62. The p62 protein has a C-terminal UBA domain that binds to ubiquitylated proteins for delivery to the 26S proteasome [START_REF] Seibenhener | Sequestosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation[END_REF]. The p62 protein also belongs to a small family of proteins that have N-terminal PB1 domains that can either selfassociate to give oligomeric signaling platforms or bind to other proteins with PB1 domains to form linked functional complexes in the cell. The MEK5 kinase has an N-terminal PB1 domain and binds to p62 [START_REF] Moscat | Signal integration and diversification through the p62 scaffold protein[END_REF][START_REF] Moscat | Cell signaling and function organized by PB1 domain interactions[END_REF].Therefore, ubiquitylation of proteins will cause binding of p62 and in explaining why p62 is in inclusions in chronic neurodegenerative disease [START_REF] Bjorkoy | p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death[END_REF][START_REF] Komatsu | Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagydeficient mice[END_REF].

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The connection between the UPS and chronic neurodegenerative diseases was enhanced by identification of the parkin gene which is mutated in autosomal recessive juvenile onset Parkinson's disease.

Parkin is a ubiquitin protein ligase [START_REF] Kitada | Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism[END_REF]. Normal parkin accumulates in Lewy bodies in the disease [START_REF] Junn | Parkin accumulation in aggresomes due to proteasome impairment[END_REF]. Interestingly, Lewy bodies do not form when parkin is mutated suggesting that the ligase has a direct role in Lewy body formation [START_REF] Kitada | Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism[END_REF]. The connection between the UPS and chronic neurodegenerative disease is also shown by mutations of a deubiquityling enzyme UCHL-1 (PGP9.5) in a small group of patients with familial Parkinson's disease [START_REF] Zhang | Association between a polymorphism of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) gene and sporadic Parkinson[END_REF]. It is possible that Parkin and UCHL-1 are important upstream players in synucleinopathies. Genome-wide linkage analysis of a pedigree of parkinsonian-pyramidal syndrome has

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shown linkage to chromosome 22 and sequencing of candidate genes showed disease-associated homozygous variation in an F-box protein, which is a substrate-receptor for a ubiquitin protein ligase [START_REF] Shojaee | Genome-wide linkage analysis of a Parkinsonian-pyramidal syndrome pedigree by 500 K SNP arrays[END_REF]. Given the importance of the UPS for neuronal development and homeostasis, it is not surprising that mutations in genes for proteins of the UPS cause chronic neurodegenerative disease. Neurological abnormalities are seen in a variety of transgenic mouse models with deficits in the UPS [START_REF] Van Tijn | The neuronal ubiquitin-proteasome system: murine models and their neurological phenotype[END_REF].

Whereas genetics identifies mutant proteins associated with disease it is more difficult to prove the role of a pathway in disease.

Some indirect evidence for malfunction of the proteasome in chronic neurodegenerative disease comes from the observation that proteasomal catalytic activity is reduced in the substantia nigra in Parkinson's disease [START_REF] Mcnaught | Proteasomal function is impaired in substantia nigra in Parkinson's disease[END_REF]. Similarly, the ubiquitin proteasome system appears impaired in the ageing brain [START_REF] Gray | Ubiquitin, proteasomes, and the aging brain[END_REF] and particularly so in Huntington's disease [START_REF] Bennett | Global changes to the ubiquitin system in Huntington's disease[END_REF],

Creutzfeld-Jakob disease, Alzheimer's disease [START_REF] Adori | The ubiquitin-proteasome system in Creutzfeldt-Jakob and Alzheimer disease: intracellular redistribution of components correlates with neuronal vulnerability[END_REF][START_REF] Lam | Inhibition of the ubiquitin-proteasome system in Alzheimer's disease[END_REF], as well as in brain ischaemia [START_REF] Ge | Protein aggregation and proteasome dysfunction after brain ischemia[END_REF][START_REF] Yao | Alterations of cerebral cortex and hippocampal proteasome subunit expression and function in a traumatic brain injury rat model[END_REF] The systemic administration of proteasomal inhibitors has been reported to cause Parkinson-like neuropathological changes, including the formation of Lewy-like inclusions in rodents [START_REF] Mcnaught | Proteasome inhibition causes nigral degeneration with inclusion bodies in rats[END_REF]. However, these observations are controversial with some laboratories being unable to reproduce the original findings [START_REF] Bove | Proteasome inhibition and Parkinson's disease modeling[END_REF][START_REF] Mcnaught | Proteasome inhibitor-induced model of Parkinson's disease[END_REF][START_REF] Manning-Bog | Lack of nigrostriatal pathology in a rat model of proteasome inhibition[END_REF][START_REF] Schapira | Proteasomal inhibition causes loss of nigral tyrosine hydroxylase neurons[END_REF]. Interestingly, α-synuclein protofilaments, but not monomers or dimers, inhibit the catalytic activity of the 26S proteasome in vitro [START_REF] Zhang | Alpha-synuclein protofibrils inhibit 26S proteasome-mediated protein degradation understanding the cytotoxicity of protein protofibrils in neurodegenerative diseases pathogenesis[END_REF].

In summary, the neuropathological insight and conclusions drawn from these findings pertaining to chronic neurodegenerative disease are;

• In neurodegenerative disease neuronal inclusions contain different proteins

• Inclusions have a primary role in disease

• Overexpression of proteins found in inclusions in mouse models may mimic disease (or overexpression of extracellular amyloid)

• The majority of intraneuronal inclusions contain ubiquitylated proteins

• Genetic insight into human neurodegenerative disease supports involvement of the UPS

• Impairment of the UPS may cause disease

• The role of the UPS is uncertain, e.g. is the UPS the primary response to protein aggregation (cf autophagy) and is the system cytoprotective?

There is clearly a need for the genetic proof or otherwise of the importance of the UPS in the chronic neurodegenerative diseases.

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16

Proteasomal Gene targeting

A decade of transgenesis in which disease-associated proteins are over-expressed singly or together in the brain has produced some, but not all features of human disease: neurodegeneration has been conspicuously absent in models of Alzheimer's disease [START_REF] Mcgowan | A decade of modeling Alzheimer's disease in transgenic mice[END_REF] and

Parkinson's disease [START_REF] Melrose | Parkinson's disease: a rethink of rodent models[END_REF]. For example, mice overexpressing mutant human α-synuclein in the brain show loss of motor neurons in the spinal cord, but not neuronal loss in the brain [START_REF] Gallardo | A molecular pathway of neurodegeneration linking alpha-synuclein to ApoE and Abeta peptides[END_REF]. These findings have been rationalized by the notion that mice are short lived animals (longevity of 2-3 years), whereas human disease occurs much later in life. In idiopathic cases, usually in the fifth to eight decades. However, plaques can form remarkably rapidly, within 24h, in a transgenic amyloid overexpressing mouse model [START_REF] Meyer-Luehmann | Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease[END_REF]. In general, the prevailing view is that mice are not men and may be of limited value in seeking the molecular key to chronic neurodegenerative disease [START_REF] Mcgowan | A decade of modeling Alzheimer's disease in transgenic mice[END_REF][START_REF] Melrose | Parkinson's disease: a rethink of rodent models[END_REF].

The alternative to transgenic over-expression of disease-related proteins is gene targeting, where candidate genes are deleted to see if neurodegeneration ensues. The targeted deletion of two autophagyrelated genes in the brain results in neurodegeneration, but not the neuropathological features of human disease [START_REF] Komatsu | Loss of autophagy in the central nervous system causes neurodegeneration in mice[END_REF][START_REF] Hara | Suppression of basal autophagy in neural cells causes neurodegeneration in mice[END_REF]. Clearly, as expected, genes for proteins involved in autophagic protein degradation are important for neuronal homeostasis and when deleted result in

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neurodegenerative disease. Such models can be further refined by conditional gene targeting, gene ablation in specific regions of the brain to reflect selective vulnerability seen in neurological illnesses. There is, of course, the dilemma of the choice of the appropriate gene for deletion.

Based on the finding of abundant ubiquitylated proteins in inclusions in all the human chronic neurodegenerative diseases, it was logical to hypothesise that conditional deletion of a proteasomal protein in the brain may recapitulate the neuropathological and behavioral abnormalities of different human neurodegenerative diseases. In addition, it is wiser to delete a 19S regulator gene than a 20S core gene, in order to be able to examine a corollary hypothesis that 20S catalytic activity may be sufficient to prevent neurodegeneration. In vitro, 20S

proteasomes have been shown to degrade α-synuclein and tau [START_REF] Tofaris | alpha-Synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome[END_REF][START_REF] David | Proteasomal degradation of tau protein[END_REF].

Affecting a 19S proteasomal gene would demonstrate if ubiquitylated proteins could accumulate in the cell and be followed by recapitulation of any of the neuronal inclusions found in human disease. The six proteasomal ATPases are non-redundant [START_REF] Seeger | Characteristics of 26 S proteases from fission yeast mutants, which arrest in mitosis[END_REF] and putative hexameric ring formation will not occur in neurons lacking an individual ATPase. An attractive candidate gene is the Psmc1 ATPase gene (S4/rpt2), since studies in yeast and in vitro show that this ATPase has a central role in connecting protein unfolding to the proteolytic activity of the 20S proteasome core [START_REF] Rubin | Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome[END_REF][START_REF] Smith | Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry[END_REF]. Conditional targeted deletion of an ATPase

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18 gene in mouse brain might be expected to recapitulate the elusive neuronal cell death, which is so prevalent in human chronic neurodegenerative disease and, in the process, produce inclusions characteristic of human disease.

A formidable and technically successful method of gene deletion involves the use of the Cre/loxP system [START_REF] Kos | Cre/loxP system for generating tissue-specific knockout mouse models[END_REF]. In this approach, the gene of interest is modified by the insertion of intronic loxP sequences, such that in the presence of Cre recombinase, the loxP flanked region is deleted. Once mice carrying the "floxed" target gene have been established these are mated with appropriate "Cre-deletor" mice to cause spatiotemporal ablation of the gene of interest. In theory, the gene of interest can be deleted in any cell type in the body of the "floxed" mice with an appropriate cell-specific Cre recombinase.

Deletor strains of mice are available so that ablation of the Psmc1 gene will only occur in catecholaminergic neurons [START_REF] Lindeberg | Transgenic expression of Cre-recombinase from the tyrosine hydroxylase locus[END_REF], including dopaminergic neurons of the substantia nigra, or in the forebrain (cortex, hippocampus, striatum and amygdala) [START_REF] Tsien | Subregion-and cell type-restricted gene knockout in mouse brain[END_REF][START_REF] Lindeberg | Timing the doxycycline yields different patterns of genomic recombination in brain neurons with a new inducible Cre transgene[END_REF]. Deletion of the gene in the substantia nigra may cause the neuropathology of Parkinson's disease, while deletion of the gene in the forebrain may cause an Alzheimer-related disorder e.g. dementia with Lewy bodies.

This experimental approach has been successful [START_REF] Bedford | Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies[END_REF]. Crosses of "floxed" Psmc1 mice with tyrosine hydroxylase (TH)-driven Cre disease [START_REF] Hoglinger | The pRb/E2F cell-cycle pathway mediates cell death in Parkinson's disease[END_REF] and in models of neuronal loss in the brain [START_REF] Herrup | Cell cycle regulation in the postmitotic neuron: oxymoron or new biology?[END_REF].

Using a calcium calmodulin-dependent protein kinase IIa (CamKIIa)-expressing Cre recombinase mouse results in Psmc1 inactivation in forebrain regions. Neuronal cell death and Lewy-like bodies characteristic of dementia with Lewy bodies are found in the forebrain, together with some behavioral abnormalities of the disease [START_REF] Bedford | Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies[END_REF]. The neuropathology is relatively rapid in these mouse models, within 12 weeks. Chronic neurodegeneration may occur in old people but can be reproduced in a matter of weeks in these models [START_REF] Bedford | Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies[END_REF].

The sequence of events in the "Lewy mouse" begins with the accumulation of ubiquitylated proteins in neurons following 26S The occurrence of α-synuclein in Lewy-like bodies after 26S proteasome deletion is interesting and suggests that α-synuclein is not only involved in phospholipid and synaptic vesicle function [START_REF] Jenco | Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by alpha-and beta-synucleins[END_REF], but may also have a primary role in Lewy body formation. This would explain why α-synuclein is present in Lewy bodies. An accepted notion [START_REF] Spillantini | alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies[END_REF] is that the aggregate-prone α-synuclein (and mutant forms) is deposited in Lewy bodies deliberately as a sequestration pathway to remove the protein from the cytoplasm (in addition to being degraded by the UPS and autophagy). However, if α-synuclein is necessary for Lewy body formation and the Lewy body is a specific cytoprotective response, then this gives a primary and functional reason for the incorporation of αsynuclein (and mutant forms?) into Lewy bodies.

Ultrastructural observations in the "Lewy mice" show that the perinuclear Lewy-like bodies are built around a core of mitochondria, surrounded by filamentous material with a periphery of membranes and autophagolysomes. This picture is the same as in Epstein-Barr virus transformed lymphoblastoid cells expressing a small number of viral proteins including the latent membrane protein [START_REF] Laszlo | The latent membrane protein-1 in Epstein-Barr virustransformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organizing centre[END_REF]. In the mouse, inclusions containing mitochondria may be the equivalent of "pale bodies" seen in human disease, which contain mitochondria and may be

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an early form of Lewy body [START_REF] Bedford | Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies[END_REF][START_REF] Gai | In situ and in vitro study of colocalization and segregation of alpha-synuclein, ubiquitin, and lipids in Lewy bodies[END_REF]. In the mouse models, early neuronal death will prevent the formation of mature Lewy bodies, containing predominately aggregated filamentous α-synuclein [START_REF] Bedford | Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies[END_REF]. There is increasing evidence that the pathologies associated with dementia with Lewy bodies, Parkinson's disease and Alzheimer's disease co-exist in the ageing brain to varying degrees and at the same time [START_REF] Burns | Clinical practice with anti-dementia drugs: consensus statement from British Association for Pyschopharamacology[END_REF].

The

For example, most patients with Parkinson's disease eventually become demented with extensive cortical and brain stem Lewy bodies [START_REF] Aarsland | Prevelence and characteristics of dementia in Parkinson's disease[END_REF].

Patients with Alzheimer's disease have Lewy body pathology, with as many as 60% having Lewy bodies in the amygdala [START_REF] Uchikado | Alzheimer disease with amygdala Lewy bodies: a distinct form of alpha-synucleinopathy[END_REF]. Clinically, the diseases are seen to be heterogeneous, a mixture of pathologies,

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23 neurochemical deficits and cognitive decline, making treatment through neurochemical replacement or receptor agonists difficult. There are only small "windows of opportunity" for treatment with existing drugs and complex responses depending on the pathological nature and extent of disease in the brains of individual patients [START_REF] Burns | Clinical practice with anti-dementia drugs: consensus statement from British Association for Pyschopharamacology[END_REF]. Hence the need for new approaches that will come from studies on models the "Lewy" mice. What is already clear from these mice is that neuronal death can be triggered by depletion of 26S proteasomes in the absence of neurofibrillary tangles or plaque amyloid. Yet, the opposite is not true in the transgenic overexpressing models, abundant amyloid and/or tangles or α-synuclein deposits do not give rise to rapid or extensive neuronal death in the brain [START_REF] Mcgowan | A decade of modeling Alzheimer's disease in transgenic mice[END_REF][START_REF] Gallardo | A molecular pathway of neurodegeneration linking alpha-synuclein to ApoE and Abeta peptides[END_REF].

One scenario is that neurofibrillary tangles and plaque amyloid increase in amounts in the ageing brain without causing neuronal death or cognitive impairment. If, however, there is concurrent proteasomal malfunction or impairment in the same brains, further pathology will occur with Lewy body formation, followed by extensive neuronal death and irreversible cognitive decline and florid dementing illness. This "UPS critical failure" model could explain the transition from normal aging to disease. Whether 26S proteasomal dysfunction always occurs in human chronic neurodegenerative disease is not proven: that 26S proteasomal depletion causes neurodegeneration in the mouse is proven [START_REF] Bedford | Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies[END_REF]. aggresomal proteins [START_REF] Wigley | Dynamic association of proteasomal machinery with the centrosome[END_REF]. When the UPS was in its infancy, protein microinjection studies into several tissue culture cell types showed that injected proteins were transported to a perinuclear "protein sequestration site associated with the microtubule organizing centre for protein degradation by autophagy" [START_REF] Earl | A putative proteinsequestration site involving intermediate filaments for protein degradation by autophagy. Studies with transplanted Sendai viral envelope proteins in HTC cells[END_REF][START_REF] Doherty | A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells[END_REF], like in EBV-transformed lymphoblastoid cells [START_REF] Laszlo | The latent membrane protein-1 in Epstein-Barr virustransformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organizing centre[END_REF]. Protein overload, whether in the cytosol or plasma membrane, sent proteins to this site quickly followed by slow autophagolysosomal degradation of the proteins (24-96 hours). In the substantia nigra, ablation of the expression of the proteasomal Psmc1 gene results in relatively rapid Lewy body formation (within 2 weeks).

In the 26S proteasome deficient models, electron microscopy shows that Lewy-like bodies are filled with mitochondria. In the EBVtransformed cells mitochondria were also a prominent feature of the inclusions [START_REF] Laszlo | The latent membrane protein-1 in Epstein-Barr virustransformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organizing centre[END_REF]. There have been few reports of mitochondria in Lewy bodies [START_REF] Gai | In situ and in vitro study of colocalization and segregation of alpha-synuclein, ubiquitin, and lipids in Lewy bodies[END_REF][START_REF] Hayashida | An early cytoplasmic change before Lewy body maturation: an ultrastructural study of the substantia nigra from an autopsy case of juvenile parkinsonism[END_REF], but the mitochondrial inner-membrane PINK kinase is found experimentally in Lewy bodies [START_REF] Abou-Sleiman | Expanding insights of mitochondrial dysfunction in Parkinson's disease[END_REF]. The PINK kinase is mutated in familial Parkinson's disease and appears to act in mitochondria upstream of mitochondrial parkin [START_REF] Park | Mitochondrial dysfunction in Drosophila PINK1 mutanrts is complimented by parkin[END_REF][START_REF] Clark | Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin[END_REF]. The PINK kinase and parkin seem to control mitochondrial fission [START_REF] Poole | The PINK1/Parkin pathway regulates mitochondrial morphology[END_REF]. The proportion of neuronal mitochondria that are incorporated into Lewy bodies is unknown.

However, mitochondria have a central role in chronic neurodegenerative diseases, most strikingly in Parkinson's disease [START_REF] Abou-Sleiman | Expanding insights of mitochondrial dysfunction in Parkinson's disease[END_REF]. If Lewy bodies

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prolong the life of the neuron then could the ability to form Lewy bodies be dependent on normal mitochondria? Lewy bodies generally do not form in Parkinson's disease associated with parkin mutations [START_REF] Giasson | Parkin and the molecular pathways of Parkinson's disease[END_REF].

Mitochondria are integral to the intrinsic pathway of apoptosis by releasing cytochrome c, triggering apoptosome formation and the activation of caspases 9 and 3 [START_REF] Tanaka | Inducible expression of mutant alpha-synuclein decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis[END_REF]. It is tempting to speculate that if mitochondria in early forms (pale bodies) of Lewy bodies [START_REF] Dale | Relationships between Lewy bodies and pale bodies in Parkinson's disease[END_REF] (which may be in the early inclusions to provide localized ATP for the biogenesis of early forms of Lewy bodies) are not subsequently removed these mitochondria may act as apoptotic "detonators" which trigger apoptosis by mechanisms including the localized release of cytochrome c. Surviving neurons, in which mitochondria are removed from maturing inclusions, will escape such a process and display the mature Lewy bodies.

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A unifying intraneuronal mechanism involving 26S proteasome malfunction and cell death in neurodegeneration

Critical failure of the 26S proteasome may be at the heart of an escalating process of neurodegeneration, in which adverse stimuli combine to increase the magnitude of regional neurodegeneration in the brain. Upstream events involving parkin and a-synuclein may also be major players in the disease process.

What now in 26S proteasome-depleted animals?

There are several predictions and hypotheses that can now be made as a result of the generation of the Lewy mouse and ensuing recapitulation of the pathology of human neurodegenerative disease including;

• The α-synuclein protein is necessary for the Lewy-like response in neurons

•

  have diagnostic methods to determine which are present. The historical diagnosis and division of the major chronic neurodegenerative diseases into entities such as Parkinson's disease and Alzheimer's disease could A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6 be unhelpful because of the overlapping pathologies found in many patients. It is possibly more useful to think of co-exisiting neurofibrillary tangles, amyloid deposits, Lewy bodies and neuronal loss together with extensive gliosis (glial cells proliferate in association with advancing pathology in the brain).

  The signaling pathways (neuronal and/or glial) involved in aggresome formation and neuronal survival or cell death are currently incompletely characterised. Ubiquitylation has been shown to be part of a signaling pathway in afflicted neurons. Protein aggregate formation in tissue culture cells (not caused by disease-associated aggregate-prone proteins) causes transient expression of the MEK5 gene. The MEK5 kinase has only one known A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 12 downstream substrate ERK5. This pathway has important developmental

A

  C C E P T E D M A N U S C R I P T recombinase mice leads to the death of nigrostriatal neurons and the appearance of Lewy-like bodies in the substantia nigra, pathognomic characteristics of Parkinson's disease. Nissl staining shows loss of neuronal RNA-containing rough endoplasmic reticulum in surviving neurons. Statistical evidence for enlarged neuronal nuclei (nucleomegaly) perhaps suggests that S4-depleted neurons instigate the cell cycle as part of the neuronal death process (Bedford & Mayer, unpublished). This has been demonstrated in the nigra in Parkinson's

  proteasomal depletion. Selective 26S depletion, with retention of 20S activity, was confirmed by glycerol gradient analyses. The appearance of α-synuclein, ubiquitin and p62 positive Lewy-like inclusions and A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 20 progressive neurodegeneration and gliosis follow rapidly. Neuronal death is indicative of apoptotic mechanisms. Ablation of the ATPase gene in catecholaminergic neurons occurs throughout the autonomic nervous system and in the neuroendocrine adrenal medulla, resulting in the premature death of these animals. The neurodegeneration in the nigrostriatal pathway is accompanied by a corresponding loss of catecholamines, including dopamine in appropriate regions of the brain, but not indolamines. Following forebrain-specific deletion of PSMC1, comprehensive analyses indicate progressive behavioral and memory deficits, including a decline in visuospatial memory functions that may mimic those seen in dementia with Lewy bodies. Details of these behavioural abnormalities can be found in the supplementary information of Bedford et al. (2008). These neuropathological changes and behavioral abnormalities occur after deletion of 26S proteasomes without any evidence for the accumulation of amyloid plaques or neurofibrillary tangles. Therefore, extensive neuronal cell death and Lewy-like body deposition can occur in the brain in the absence of the neuropathological hallmarks of Alzheimer's disease. The immunohistochemical and morphological features of neurons in 26S proteasome-depleted mice are indicative of an "aggresomal" pathway of neurodegeneration, involving α-synuclein, protein ubiquitylation, p62 and other proteins together as A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 21 seen in human disease. Additionally, mitochondria are a prominent feature of the inclusions in surviving neurons.

  26S proteasome-depleted models for understanding and treatment of Parkinson's disease and Dementia with Lewy bodies The outcomes of conditional Psmc1 gene deletion in the nigrostriatal pathway and forebrain support the hypothesis that the 26S proteasome is essential for normal neuronal homeostasis. Depletion of 26S proteasomes will cause chronic neurodegenerative disease, resembling both Parkinson's disease and dementia with Lewy bodies [81]. Additionally, residual 20S proteasomes are insufficient for neuronal survival. The findings have considerable implications for human diseases, which are generally thought of as diseases of old-age with neuropathological changes occurring over periods of years.

  It is not yet clear what the exact role of the neuronal Lewy bodyis in this situation. Opinion on the role of the Lewy body swings like a pendulum from protector to villain. The current position of the pendulum favours a cytoprotective role, particularly if the Lewy body is seen as an "aggresome"[START_REF] Arrasate | Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death[END_REF]. There are only three known mechanisms for the disposal of problem proteins in the cell: degradation by the UPS, degradation by autophagy or incorporation into aggresomes[START_REF] Welchman | Ubiquitin and ubiquitin-like proteins as multifunctional signals[END_REF].Degradation by the endosome-lysosome system is also available for proteins internalized from the cell surface[START_REF] Mayer | The meteoric rise of regulated intracellular proteolysis[END_REF]. It is now clear from gene targeting studies that ablation of autophagy genes in the brain, while leading to neurodegeneration with accumulation of ubiquitylated proteins, does not cause features of human disease such as Lewy bodies[START_REF] Komatsu | Loss of autophagy in the central nervous system causes neurodegeneration in mice[END_REF][START_REF] Hara | Suppression of basal autophagy in neural cells causes neurodegeneration in mice[END_REF]. Ablation of the proteasomal PSMC1 ATPase does recapitulate human neuropathology including Lewy-like bodies. In the 26S proteasome-deficient neurons proteins accumulate in perinuclear structures most likely built around the microtubule organizing centre (centrosome) like in EBV-virus transformed cells[START_REF] Laszlo | The latent membrane protein-1 in Epstein-Barr virustransformed lymphoblastoid cells is found with ubiquitin-protein conjugates and heat-shock protein 70 in lysosomes oriented around the microtubule organizing centre[END_REF]. These structures are very similar to the aggresomes seen in different experimental models with aggregate-prone proteins. For operational purposes the Lewy body is almost certainly derived from a neuronal aggresome. Experimental aggresomes contain vimentin filaments and components of the UPS as well as peripheral autophagolysosomes presumably trying to degrade the A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 25

Fig. 2 (

 2 Fig. 2 (escalating cascade of neurodegeneration)This process would be initiated by the accumulation of different types of intraneuronal proteins, triggering chaperone, UPS, autophagic and cell survival responses followed by inclusion body biogenesis. As a consequence of 26S proteasome inhibition by aggregate-prone proteins[START_REF] Bennett | Global changes to the ubiquitin system in Huntington's disease[END_REF][START_REF] Bence | Impairment of the ubiquitinproteasome system by protein aggregation[END_REF][START_REF] Bennett | Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation[END_REF], or as shown genetically in the mouse models, 26S proteasome malfunction or depletion triggers neuronal death, which, because of the interacting neuronal pathways and projections, escalates to result in the extensive regional neurodegeneration characteristic of human disease.Clearly, the 26S proteasome is central for neuronal survival and depletion of 26S proteasomes by ablation of the PSMC1 ATPase in appropriate specific regions of the brain recapitulates neuropathological

•

  Early involvement of mitochondria is a key feature of the Lewy body response Failure to abrogate the causative stimulus for Lewy body formation leads to mitochondrial-dependent apoptosis: this is a critical factor during Lewy body biogenesis and equates with the "pale body" seen in human disease A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Acknowledgements

The Wellcome Trust, BBSRC, Alzheimer Research Trust, Parkinson's Disease Society and Neuroscience Support Group at the Queen's Medical Centre have sponsored some of the work described in this article.