Simon Paine

Depletion of 26S Proteasomes in Mouse Brain Neurons Causes Neurodegeneration and Lewy-Like Inclusions Resembling Human Pale Bodies

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinsons disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and α-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinsons disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Assembly, structure, and function of the 26S proteasome

The 26S proteasome is a large multiprotein complex involved in the regulated degradation of ubiquitinated proteins in the cell. The 26S proteasome has been shown to control an increasing number of essential biochemical mechanisms of the cellular lifecycle including DNA synthesis, repair, transcription, translation, and cell signal transduction. Concurrently, it is increasingly seen that malfunction of the ubiquitin proteasome system contributes to the pathogenesis of disease. The recent identification of four molecular chaperones, in addition to five previously identified chaperones, have provided mechanistic insight into how this cellular megastructure is assembled in the cell. These data, together with new insights into the structure and function of the proteasome, provide a much better understanding of this complex protease.

Astrovirus VA1/HMO-C: An Increasingly Recognized Neurotropic Pathogen in Immunocompromised Patients

Brain biopsy from a child with unknown cause of encephalopathy was deep-sequenced. Astrovirus VA1/HMO-C was identified, highly divergent from human astroviruses and 95% identical to astrovirus previously associated with encephalitis. Findings suggest astrovirus VA1/HMO-C is an under-recognized cause of viral encephalitis.

mTOR-dependent abnormalities in autophagy characterize human malformations of cortical development: evidence from focal cortical dysplasia and tuberous sclerosis

Focal cortical dysplasia (FCD) is a localized malformation of cortical development and is the commonest cause of severe childhood epilepsy in surgical practice. Children with FCD are severely disabled by their epilepsy, presenting with frequent seizures early in life. The commonest form of FCD in children is characterized by the presence of an abnormal population of cells, known as balloon cells. Similar pathological changes are seen in the cortical malformations that characterize patients with tuberous sclerosis complex (TSC). However, the cellular and molecular mechanisms that underlie the malformations of FCD and TSC are not well understood. We provide evidence for a defect in autophagy in FCD and TSC. We have found that balloon cells contain vacuoles that include components of the autophagy pathway. Specifically, we show that balloon cells contain prominent lysosomes by electron microscopy, immunohistochemistry for LAMP1 and LAMP2, LysoTracker labelling and enzyme histochemistry for acid phosphatase. Furthermore, we found that balloon cells contain components of the ATG pathway and that there is cytoplasmic accumulation of the regulator of autophagy, DOR. Most importantly we found that there is abnormal accumulation of the autophagy cargo protein, p62. We show that this defect in autophagy can be, in part, reversed in vitro by inhibition of the mammalian target of rapamycin (mTOR) suggesting that abnormal activation of mTOR may contribute directly to a defect in autophagy in FCD and TSC.

Review: the ubiquitin-proteasome system: contributions to cell death or survival in neurodegeneration.

N. Rogers, S. Paine, L. Bedford and R. Layfield (2010) Neuropathology and Applied Neurobiology36, 113–124
The ubiquitin‐proteasome system: contributions to cell death or survival in neurodegeneration

Is malfunction of the ubiquitin proteasome system the primary cause of α-synucleinopathies and other chronic human neurodegenerative disease?

Neuropathological investigations have identified major hallmarks of chronic neurodegenerative disease. These include protein aggregates called Lewy bodies in dementia with Lewy bodies and Parkinsons disease. Mutations in the alpha-synuclein gene have been found in familial disease and this has led to intense focused research in vitro and in transgenic animals to mimic and understand Parkinsons disease. A decade of transgenesis has lead to overexpression of wild type and mutated alpha-synuclein, but without faithful reproduction of human neuropathology and movement disorder. In particular, widespread regional neuronal cell death in the substantia nigra associated with human disease has not been described. The intraneuronal protein aggregates (inclusions) in all of the human chronic neurodegenerative diseases contain ubiquitylated proteins. There could be several reasons for the accumulation of ubiquitylated proteins, including malfunction of the ubiquitin proteasome system (UPS). This hypothesis has been genetically tested in mice by conditional deletion of a proteasomal regulatory ATPase gene. The consequences of gene ablation in the forebrain include extensive neuronal death and the production of Lewy-like bodies containing ubiquitylated proteins as in dementia with Lewy bodies. Gene deletion in catecholaminergic neurons, including in the substantia nigra, recapitulates the neuropathology of Parkinsons disease.

Clinical and neuropathological features of X-linked spinal muscular atrophy (SMAX2) associated with a novel mutation in the UBA1 gene

Infantile-onset X-linked spinal muscular atrophy (SMAX2) is a rare lethal disorder linked to mutations in the UBA1 (previously UBE1) gene, encoding ubiquitin-activating enzyme 1 that has an important role in the ubiquitin-proteasome pathway. Published pathological reports are scarce. Here we report a male infant who presented from birth with predominantly truncal hypotonia following an antenatal history of reduced fetal movements. He had a myopathic face, profound weakness, multiple contractures and areflexia. Creatine kinase was moderately raised. Brain MRI showed non-specific symmetrical periventricular white matter changes. Neurophysiology revealed evidence of motor and sensory involvement and muscle biopsy showed marked inflammatory changes with subtle features suggestive of acute denervation. UBA1 sequencing revealed a novel hemizygous missense mutation (c.1670A>T; p.Glu557Val). He died from progressive respiratory failure at 4 months. On post mortem assessment, in addition to severe ventral motor neuron pathology, there was widespread involvement of the sensory system, as well as developmental and degenerative cerebellar abnormalities. In contrast to typical SMN1-associated SMA, the thalamus was unaffected. These findings indicate that SMAX2 is more accurately classified as a motor sensory neuronopathy rather than a pure anterior horn cell disorder. Ubiquitin-proteasome pathway defects may not only cause neurodegeneration but also affect normal neuronal development.

Immunoreactivity to Lys63-linked polyubiquitin is a feature of neurodegeneration

The major human neurodegenerative diseases are characterised by ubiquitin-positive intraneuronal inclusions, however the precise nature of the ubiquitin modifications in these structures is unclear. Using a monoclonal antibody specific for Lys63-linked polyubiquitin we have performed the first immunohistochemical analysis of linkage-specific ubiquitination in vivo associated with neurodegeneration. Immunoreactivity was detected within the pathological lesions of Alzheimers, Huntingtons and Parkinsons disease brains, although staining of Lewy bodies in the substantia nigra in Parkinsons disease was rare, indicating a selective involvement of Lys63-linked polyubiquitin in inclusion biogenesis in this disorder. Immunoreactivity was also a feature in neurons of proteasome-depleted mice, suggesting a proteasomal contribution to the degradation of Lys63-linked polyubiquitinated proteins in vivo.

Review: Neuropathological features of unexplained sudden unexpected death in infancy: current evidence and controversies

Sudden infant death syndrome (SIDS) is a leading cause of postneonatal infant death in the developed world. The cause of SIDS is unknown but several hypotheses have been proposed, including the ‘triple risk hypothesis’, which predicts that foetal development of infants who subsequently succumb to SIDS is abnormal, leaving them unable to respond appropriately to stressors. Consistent with this hypothesis, a large number of studies have reported changes in the brain in SIDS. However, on nearly every subject, the reported findings vary widely between studies. Inconsistencies in the definitions of SIDS used and in control group selection are likely to underlie much of this variability. Therefore, in our analysis, we have included only those studies that met simple criteria for both the definition of SIDS and the control group. Of the 153 studies retrieved by our review of the literature, 42 (27%) met these criteria. Foremost among the findings reported by these studies are abnormalities of the brain stem, in particular brain stem gliosis and defects of neurotransmission in the medulla. However, these studies have not identified what could be considered in diagnostic terms a causative structural or biochemical abnormality for use in routine clinical practice. An assessment of changes in the architecture and composition of brain regions and changes in neurotransmission in multiple systems in a single, large cohort of well‐ and consistently characterized infants dying suddenly of a range of causes is needed before the inter‐relation of these different features can be appreciated.

The UPS and autophagy in chronic neurodegenerative disease Six of one and half a dozen of the other-or not?

In the past twenty years, evidence has accumulated to show that ubiquitinated proteins are a consistent feature of the intraneuronal protein aggregates (inclusions) that characterize chronic neurodegenerative disease. These findings may indicate that age-related dysfunction of the 26S proteasome may be central to disease pathogenesis. The aggregate-prone proteins can also be eliminated by autophagy. We have used the Cre-recombinase/loxP genetic approach to ablate the proteasomal psmc1 ATPase gene and deplete 26S proteasomes in neurons in different regions of the brain to mimic neurodegeneration. Deletion of the gene in dopaminergic neurons in the substantia nigra generates a new model of Parkinson’s disease. Ablation of the gene in the forebrain creates the first model of dementia with Lewy bodies. In both neuroanatomical regions, gene ablation causes the formation of Lewy-like inclusions together with extensive neurodegeneration. There is some evidence for neuronal autophagy in areas adjacent to inclusions. The models indicate that neuronal loss in neurodegenerative diseases can be attributed to proteasomal malfunction accompanied by Lewy-like inclusions as seen in dementia with Lewy bodies and Parkinson’s disease.