Proteomics in neurodegenerative diseases: Methods for obtaining a closer look at the neuronal proteome

Plum, Sarah; Steinbach, Simone; Abel, Laura; Marcus, Katrin; Helling, Stefan; May, Caroline

doi:10.1002/prca.201400030

Cited by 12 publications

(11 citation statements)

References 279 publications

(322 reference statements)

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“…In contrast to PMCA2, the Ca 2+ -binding protein calretinin [ 84 ] was shown to be significantly increased in the mdx - 4cv brain (Table 1 ), possibly compensating abnormal calcium fluxes within specific neurons of the central nervous system. Interesting proteomic biomarker candidates with a differential expression pattern are the neuron-specific enzyme ubiquitin carboxyl-terminal hydrolase isozyme L1, the dendritic spine protein drebrin, the cytomatrix protein bassoon of the nerve terminal active zone, and the synapse-associated protein SAP97, as well as the nervous system-specific proteins syntaxin-1B and syntaxin-binding protein 1 [ 85 ].…”

Section: Resultsmentioning

confidence: 99%

Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy

et al. 2015

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BackgroundX-linked muscular dystrophy is a primary disease of the neuromuscular system. Primary abnormalities in the Dmd gene result in the absence of the full-length isoform of the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle wasting and cardio-respiratory complications, developmental cognitive deficits and behavioural abnormalities are clinical features of Duchenne muscular dystrophy. In order to better understand the mechanisms that underlie impaired brain functions in Duchenne patients, we have carried out a proteomic analysis of total brain extracts from the mdx-4cv mouse model of dystrophinopathy.ResultsThe comparative proteomic profiling of the mdx-4cv brain revealed a significant increase in 39 proteins and a decrease in 7 proteins. Interesting brain tissue-associated proteins with an increased concentration in the mdx-4cv animal model were represented by the glial fibrillary acidic protein GFAP, the neuronal Ca2+-binding protein calretinin, annexin AnxA5, vimentin, the neuron-specific enzyme ubiquitin carboxyl-terminal hydrolase isozyme L1, the dendritic spine protein drebrin, the cytomatrix protein bassoon of the nerve terminal active zone, and the synapse-associated protein SAP97. Decreased proteins were identified as the nervous system-specific proteins syntaxin-1B and syntaxin-binding protein 1, as well as the plasma membrane Ca2+-transporting ATPase PMCA2 that is mostly found in the brain cortex. The differential expression patterns of GFAP, vimentin, PMCA2 and AnxA5 were confirmed by immunoblotting. Increased GFAP levels were also verified by immunofluorescence microscopy.ConclusionsThe large number of mass spectrometrically identified proteins with an altered abundance suggests complex changes in the mdx-4cv brain proteome. Increased levels of the glial fibrillary acidic protein, an intermediate filament component that is uniquely associated with astrocytes in the central nervous system, imply neurodegeneration-associated astrogliosis. The up-regulation of annexin and vimentin probably represent compensatory mechanisms involved in membrane repair and cytoskeletal stabilization in the absence of brain dystrophin. Differential alterations in the Ca2+-binding protein calretinin and the Ca2+-pumping protein PMCA2 suggest altered Ca2+-handling mechanisms in the Dp427-deficient brain. In addition, the proteomic findings demonstrated metabolic adaptations and functional changes in the central nervous system from the dystrophic phenotype. Candidate proteins can now be evaluated for their suitability as proteomic biomarkers and their potential in predictive, diagnostic, prognostic and/or therapy-monitoring approaches to treat brain abnormalities in dystrophinopathies.

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Section: Resultsmentioning

confidence: 99%

Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy

et al. 2015

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“…Previous studies have only partially addressed these issues. 23 – 27 The identification of these pathways is crucial for elucidating the processes mediating neuronal survival and vulnerability during aging and PD.…”

Section: Introductionmentioning

confidence: 99%

Neuromelanin organelles are specialized autolysosomes that accumulate undegraded proteins and lipids in aging human brain and are likely involved in Parkinson’s disease

Zucca

Vanna

Cupaioli

et al. 2018

npj Parkinson's Disease

108

147

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During aging, neuronal organelles filled with neuromelanin (a dark-brown pigment) and lipid bodies accumulate in the brain, particularly in the substantia nigra, a region targeted in Parkinson’s disease. We have investigated protein and lipid systems involved in the formation of these organelles and in the synthesis of the neuromelanin of human substantia nigra. Membrane and matrix proteins characteristic of lysosomes were found in neuromelanin-containing organelles at a lower number than in typical lysosomes, indicating a reduced enzymatic activity and likely impaired capacity for lysosomal and autophagosomal fusion. The presence of proteins involved in lipid transport may explain the accumulation of lipid bodies in the organelle and the lipid component in neuromelanin structure. The major lipids observed in lipid bodies of the organelle are dolichols with lower amounts of other lipids. Proteins of aggregation and degradation pathways were present, suggesting a role for accumulation by this organelle when the ubiquitin-proteasome system is inadequate. The presence of proteins associated with aging and storage diseases may reflect impaired autophagic degradation or impaired function of lysosomal enzymes. The identification of typical autophagy proteins and double membranes demonstrates the organelle’s autophagic nature and indicates that it has engulfed neuromelanin precursors from the cytosol. Based on these data, it appears that the neuromelanin-containing organelle has a very slow turnover during the life of a neuron and represents an intracellular compartment of final destination for numerous molecules not degraded by other systems.

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“…Further, the possibility to combine LMD with other fractionation strategies could reveal a deeper insight in molecular processes. To continue with the example of substantia nigra analysis, a purification and enrichment of neuromelanin granula or mitochondria out of laser microdissected neurons (Plum et al 2014) could give a better understanding of molecular processes in cells. LMD has many advantages and is simple to execute.…”

Section: Discussionmentioning

confidence: 99%

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis

et al. 2015

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Brain function in normal aging and neurological diseases has long been a subject of interest. With current technology, it is possible to go beyond descriptive analyses to characterize brain cell populations at the molecular level. However, the brain comprises over 100 billion highly specialized cells, and it is a challenge to discriminate different cell groups for analyses. Isolating intact neurons is not feasible with traditional methods, such as tissue homogenization techniques. The advent of laser microdissection techniques promises to overcome previous limitations in the isolation of specific cells. Here, we provide a detailed protocol for isolating and analyzing neurons from postmortem human brain tissue samples. We describe a workflow for successfully freezing, sectioning and staining tissue for laser microdissection. This protocol was validated by mass spectrometric analysis. Isolated neurons can also be employed for western blotting or PCR. This protocol will enable further examinations of brain cell-specific molecular pathways and aid in elucidating distinct brain functions.

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Proteomics in neurodegenerative diseases: Methods for obtaining a closer look at the neuronal proteome

Cited by 12 publications

References 279 publications

Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy

Label-free mass spectrometric analysis reveals complex changes in the brain proteome from the mdx-4cv mouse model of Duchenne muscular dystrophy

Neuromelanin organelles are specialized autolysosomes that accumulate undegraded proteins and lipids in aging human brain and are likely involved in Parkinson’s disease

Enrichment of single neurons and defined brain regions from human brain tissue samples for subsequent proteome analysis

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