Variant biomarker discovery using mass spectrometry-based proteogenomics.

Genomic diversity plays critical roles in risk of disease pathogenesis and diagnosis. While genomic variants-including single nucleotide variants, frameshift variants, and mis-splicing isoforms-are commonly detected at the DNA or RNA level, their translated variant protein or polypeptide products are ultimately the functional units of the associated disease. These products are often released in biofluids and could be leveraged for clinical diagnosis and patient stratification. Recent emergence of integrated analysis of genomics with mass spectrometry-based proteomics for biomarker discovery, also known as proteogenomics, have significantly advanced the understanding disease risk variants, precise medicine, and biomarker discovery. In this review, we discuss variant proteins in the context of cancers and neurodegenerative diseases, outline current and emerging proteogenomic approaches for biomarker discovery, and provide a comprehensive proteogenomic strategy for detection of putative biomarker candidates in human biospecimens. This strategy can be implemented for proteogenomic studies in any field of enquiry. Our review timely addresses the need of biomarkers for aging related diseases.

The endoplasmic reticulum contributes to lysosomal tubulation/sorting driven by LRRK2.

Lysosomes are dynamic organelles that can remodel their membrane as an adaptive response to various cell signaling events including membrane damage. Recently, we have discovered that damaged lysosomes form and sort tubules into moving vesicles. We named this process LYTL for LYsosomal Tubulation/sorting driven by LRRK2, as the Parkinson's disease protein LRRK2 promotes tubulation by recruiting the motor adaptor protein JIP4 to lysosomes via phosphorylated RAB proteins. Here we use spinning-disk microscopy combined with superresolution to further characterize LYTL after membrane damage with LLOMe (l-leucyl-l-leucine methyl ester). We identified the endoplasmic reticulum (ER) colocalizing with sites of fission of lysosome-derived tubules. In addition, modifying the morphology of the ER by reducing ER tubules leads to a decrease in LYTL sorting, suggesting that contact with tubular ER is necessary for lysosomal membrane sorting. Given the central roles of LRRK2 and lysosomal biology in Parkinson's disease, these discoveries are likely relevant to disease pathology and highlight interactions between organelles in this model.

DJ-1 is not a deglycase and makes a modest contribution to cellular defense against methylglyoxal damage in neurons.

Human DJ-1 is a cytoprotective protein whose absence causes Parkinson's disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity.

BACKGROUND: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. OBJECTIVE: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. METHODS: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. RESULTS: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. CONCLUSION: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal dominant Parkinson disease (PD), while polymorphic LRRK2 variants are associated with sporadic PD. PD-linked mutations increase LRRK2 kinase activity and induce neurotoxicity in vitro and in vivo. The small GTPase Rab8a is a LRRK2 kinase substrate and is involved in receptor-mediated recycling and endocytic trafficking of transferrin, but the effect of PD-linked LRRK2 mutations on the function of Rab8a is poorly understood. Here, we show that gain-of-function mutations in LRRK2 induce sequestration of endogenous Rab8a to lysosomes in overexpression cell models, while pharmacological inhibition of LRRK2 kinase activity reverses this phenotype. Furthermore, we show that LRRK2 mutations drive association of endocytosed transferrin with Rab8a-positive lysosomes. LRRK2 has been nominated as an integral part of cellular responses downstream of proinflammatory signals and is activated in microglia in postmortem PD tissue. Here, we show that iPSC-derived microglia from patients carrying the most common LRRK2 mutation, G2019S, mistraffic transferrin to lysosomes proximal to the nucleus in proinflammatory conditions. Furthermore, G2019S knock-in mice show a significant increase in iron deposition in microglia following intrastriatal LPS injection compared to wild-type mice, accompanied by striatal accumulation of ferritin. Our data support a role of LRRK2 in modulating iron uptake and storage in response to proinflammatory stimuli in microglia.

Transcriptome analysis of collagen VI-related muscular dystrophy muscle biopsies.

OBJECTIVE: To define the transcriptomic changes responsible for the histologic alterations in skeletal muscle and their progression in collagen VI-related muscular dystrophy (COL6-RD). METHODS: COL6-RD patient muscle biopsies were stratified into three groups based on the overall level of pathologic severity considering degrees of fibrosis, muscle fiber atrophy, and fatty replacement of muscle tissue. Using microarray and RNA-Seq, we then performed global gene expression profiling on the same muscle biopsies and compared their transcriptome with age- and sex-matched controls. RESULTS: COL6-RD muscle biopsy transcriptomes as a group revealed prominent upregulation of muscle extracellular matrix component genes and the downregulation of skeletal muscle and mitochondrion-specific genes. Upregulation of the TGFß pathway was the most conspicuous change across all biopsies and was fully evident even in the mildest/earliest histological group. There was no difference in the overall transcriptional signature between the different histologic groups but polyserial analysis identified relative changes along with COL6-RD histological severity. INTERPRETATION: Overall, our study establishes the prominent dysregulation of extracellular matrix genes, TGFß signaling, and its downstream cellular pathways at the transcriptomic level in COL6-RD muscle.

Generation of iPSC line from a Parkinson patient with PARK7 mutation and CRISPR-edited Gibco human episomal iPSC line to mimic PARK7 mutation.

Mutations in the oncogene PARK7, which codes for DJ-1, have been associated with early-onset autosomal recessive Parkinson's disease (PD); however, the exact role of DJ-1 in PD remains elusive. Fibroblasts from a PD patient with a uniparental disomy, 1 bp deletion in PARK7 were reprogrammed into the induced pluripotent stem cell (iPSC) line: NIHTVBi015-A. For control purposes, CRISPR-Cas9 editing was used to mimic the mutation in the Gibco Human Episomal iPSC line: TMOi001-A is the control line (A18945) and TMOi001-A-3 is the control-edited line (2B10). All 3 lines exhibit normal karyotyping and expression of pluripotent markers: OCT4, SOX2, and NANOG. These lines provide a translational environment to study DJ-1-related function in PD.

Tackling neurodegenerative diseases with genomic engineering: A new stem cell initiative from the NIH.

The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever iPSC genome engineering project. iNDI will model more than 100 mutations associated with Alzheimer's disease and related dementias (ADRD) in isogenic iPSC lines. Resulting cell lines and phenotypic datasets will be broadly shared.

Extracellular clusterin limits the uptake of α-synuclein fibrils by murine and human astrocytes.

The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α-synuclein. Clearance of extracellular α-synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α-synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α-synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α-synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human-induced pluripotent stem cell (iPSC)-derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α-synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α-synuclein preformed fibrils (pffs) through dynamin-dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α-synuclein pffs exposure. Specifically, we found that clusterin interacts with α-synuclein pffs in the extracellular compartment and the clusterin/α-synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock-out primary astrocytes and clusterin knock-down hiPSC-derived astrocytes we observed that clusterin limits the uptake of α-synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno-associated virus- and the α-synuclein pffs- injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α-synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology.

LRRK2 mediates microglial neurotoxicity via NFATc2 in rodent models of synucleinopathies.

Synucleinopathies are neurodegenerative disorders characterized by abnormal α-synuclein deposition that include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The pathology of these conditions also includes neuronal loss and neuroinflammation. Neuron-released α-synuclein has been shown to induce neurotoxic, proinflammatory microglial responses through Toll-like receptor 2, but the molecular mechanisms involved are poorly understood. Here, we show that leucine-rich repeat kinase 2 (LRRK2) plays a critical role in the activation of microglia by extracellular α-synuclein. Exposure to α-synuclein was found to enhance LRRK2 phosphorylation and activity in mouse primary microglia. Furthermore, genetic and pharmacological inhibition of LRRK2 markedly diminished α-synuclein-mediated microglial neurotoxicity via lowering of tumor necrosis factor-α and interleukin-6 expression in mouse cultures. We determined that LRRK2 promoted a neuroinflammatory cascade by selectively phosphorylating and inducing nuclear translocation of the immune transcription factor nuclear factor of activated T cells, cytoplasmic 2 (NFATc2). NFATc2 activation was seen in patients with synucleinopathies and in a mouse model of synucleinopathy, where administration of an LRRK2 pharmacological inhibitor restored motor behavioral deficits. Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies.

Molecular mechanism of olesoxime-mediated neuroprotection through targeting α-synuclein interaction with mitochondrial VDAC.

An intrinsically disordered neuronal protein α-synuclein (αSyn) is known to cause mitochondrial dysfunction, contributing to loss of dopaminergic neurons in Parkinson's disease. Through yet poorly defined mechanisms, αSyn crosses mitochondrial outer membrane and targets respiratory complexes leading to bioenergetics defects. Here, using neuronally differentiated human cells overexpressing wild-type αSyn, we show that the major metabolite channel of the outer membrane, the voltage-dependent anion channel (VDAC), is a pathway for αSyn translocation into the mitochondria. Importantly, the neuroprotective cholesterol-like synthetic compound olesoxime inhibits this translocation. By applying complementary electrophysiological and biophysical approaches, we provide mechanistic insights into the interplay between αSyn, VDAC, and olesoxime. Our data suggest that olesoxime interacts with VDAC ß-barrel at the lipid-protein interface thus hindering αSyn translocation through the VDAC pore and affecting VDAC voltage gating. We propose that targeting αSyn translocation through VDAC could represent a key mechanism for the development of new neuroprotective strategies.

DNAJC proteins and pathways to parkinsonism.

The DNAJC protein family is a subclass of heat shock proteins that has attracted recent attention due to the identification of mutations that are linked with parkinsonism, a feature of Parkinson's disease and other neurological disorders. In this review, we discuss the current genetic and functional evidence of the association of these DNAJC proteins with disease and how mutations in these proteins may contribute to disease pathogenesis. Although DNAJC6 (Auxilin), DNAJC12, and DNAJC5 (CSPα) exhibit strong genetic association with disease, DNAJC26 (GAK), DNAJC13 (RME-8), and DNAJC10 (Erdj5) require additional evidence to definitively link reported variants to parkinsonism. Remarkably, multiple DNAJC proteins (Auxilin, GAK, RME-8, CSPα) functionally converge on pathways of synaptic trafficking and clathrin dynamics, highlighting an important role of those pathways in the pathogenesis of parkinsonism. Further research is required to define the mechanisms through which these mutations contribute to disease etiology.

Leucine-rich repeat kinase 2 controls protein kinase A activation state through phosphodiesterase 4.

BACKGROUND: Evidence indicates a cross-regulation between two kinases, leucine-rich repeat kinase 2 (LRRK2) and protein kinase A (PKA). In neurons, LRRK2 negatively regulates PKA activity in spiny projecting neurons during synaptogenesis and in response to dopamine D1 receptor activation acting as an A-anchoring kinase protein (AKAP). In microglia cells, we showed that LRRK2 kinase activity negatively regulates PKA, impacting NF-κB p50 signaling and the inflammatory response. Here, we explore the molecular mechanism underlying the functional interaction between LRRK2 and PKA in microglia. METHODS: To understand which step of PKA signaling is modulated by LRRK2, we used a combination of in vitro and ex vivo systems with hyperactive or inactive LRRK2 as well as different readouts of PKA signaling. RESULTS: We confirmed that LRRK2 kinase activity acts as a negative regulator of PKA activation state in microglia. Specifically, we found that LRRK2 controls PKA by affecting phosphodiesterase 4 (PDE4) activity, modulating cAMP degradation, content, and its dependent signaling. Moreover, we showed that LRRK2 carrying the G2019S pathological mutation downregulates PKA activation causing a reduction of PKA-mediated NF-κB inhibitory signaling, which results, in turn, in increased inflammation in LRRK2 G2019S primary microglia upon α-synuclein pre-formed fibrils priming. CONCLUSIONS: Overall, our findings indicate that LRRK2 kinase activity is a key regulator of PKA signaling and suggest PDE4 as a putative LRRK2 effector in microglia. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD.

Reduction of PINK1 or DJ-1 impair mitochondrial motility in neurites and alter ER-mitochondria contacts.

Subcellular distribution of mitochondria in neurons is crucial for meeting the energetic demands, as well as the necessity to buffer Ca2+ within the axon, dendrites and synapses. Mitochondrial impairment is an important feature of Parkinson disease (PD), in which both familial parkinsonism genes DJ-1 and PINK1 have a great impact on mitochondrial function. We used differentiated human dopaminergic neuroblastoma cell lines with stable PINK1 or DJ-1 knockdown to study live motility of mitochondria in neurites. The frequency of anterograde and retrograde mitochondrial motility was decreased in PINK1 knockdown cells and the frequency of total mitochondrial motility events was reduced in both cell lines. However, neither the distribution nor the size of mitochondria in the neurites differed from the control cells even after downregulation of the mitochondrial fission protein, Drp1. Furthermore, mitochondria from PINK1 knockdown cells, in which motility was most impaired, had increased levels of GSK3ßSer9 and higher release of mitochondrial Ca2+ when exposed to CCCP-induced mitochondrial uncoupling. Further analysis of the ER-mitochondria contacts involved in Ca2+ shuttling showed that PINK1 knockdown cells had reduced contacts between the two organelles. Our results give new insight on how PINK1 and DJ-1 influence mitochondria, thus providing clues to novel PD therapies.

Analysis of macroautophagy related proteins in G2019S LRRK2 Parkinson's disease brains with Lewy body pathology.

LRRK2, the gene encoding the multidomain kinase Leucine-Rich Repeat Kinase 2 (LRRK2), has been linked to familial and sporadic forms of Parkinson's disease (PD), as well as cancer, leprosy and Crohn's disease, establishing it as a target for discovery therapeutics. LRRK2 has been associated with a range of cellular processes, however its physiological and pathological functions remain unclear. The most prevalent LRRK2 mutations in PD have been shown to affect macroautophagy in various cellular models while a role in autophagy signalling has been recapitulated in vivo. Dysregulation of autophagy has been implicated in PD pathology, and this raises the possibility that differential autophagic activity is relevant to disease progression in PD patients carrying LRRK2 mutations. To examine the relevance of LRRK2 to the regulation of macroautophagy in a disease setting we examined the levels of autophagic markers in the basal ganglia of G2019S LRRK2 PD post-mortem tissue, in comparison to pathology-matched idiopathic PD (iPD), using immunoblotting (IB). Significantly lower levels of p62 and LAMP1 were observed in G2019S LRRK2 PD compared to iPD cases. Similarly, an increase in ULK1 was observed in iPD but was not reflected in G2019S LRRK2 PD cases. Furthermore, examination of p62 by immunohistochemistry (IH) recapitulated a distinct signature for G2019S PD. IH of LAMP1, LC3 and ULK1 broadly correlated with the IB results. Our data from a small but pathologically well-characterized cases highlights a divergence of G2019S PD carriers in terms of autophagic response in alpha-synuclein pathology affected brain regions compared to iPD.

AKT signalling selectively regulates PINK1 mitophagy in SHSY5Y cells and human iPSC-derived neurons.

The discovery of mutations within genes associated with autosomal recessive Parkinson's disease allowed for the identification of PINK1/Parkin regulated mitophagy as an important pathway for the removal of damaged mitochondria. While recent studies suggest that AKT-dependent signalling regulates Parkin recruitment to depolarised mitochondria, little is known as to whether this can also regulate PINK1 mitochondrial accumulation and downstream mitophagy. Here, we demonstrate that inhibition of AKT signalling decreases endogenous PINK1 accumulation in response to mitochondria depolarisation, subsequent Parkin recruitment, phosphorylation of ubiquitin, and ultimately mitophagy. Conversely, we show that upon stimulation of AKT signalling via insulin, the mitophagy pathway is increased in SHSY5Y cells. These data suggest that AKT signalling is an upstream regulator of PINK1 accumulation on damaged mitochondria. Importantly, we show that the AKT pathway also regulates endogenous PINK1-dependent mitophagy in human iPSC-derived neurons.

Proteomic analysis reveals co-ordinated alterations in protein synthesis and degradation pathways in LRRK2 knockout mice.

Mutations in leucine-rich repeat kinase 2 (LRRK2) segregate with familial Parkinson's disease (PD) and genetic variation around LRRK2 contributes to risk of sporadic disease. Although knockout (KO) of Lrrk2 or knock-in of pathogenic mutations into the mouse germline does not result in a PD phenotype, several defects have been reported in the kidneys of Lrrk2 KO mice. To understand LRRK2 function in vivo, we used an unbiased approach to determine which protein pathways are affected in LRRK2 KO kidneys. We nominated changes in cytoskeletal-associated proteins, lysosomal proteases, proteins involved in vesicular trafficking and in control of protein translation. Changes were not seen in mice expressing the pathogenic G2019S LRRK2 mutation. Using cultured epithelial kidney cells, we replicated the accumulation of lysosomal proteases and demonstrated changes in subcellular distribution of the cation-independent mannose-6-phosphate receptor. These results show that loss of LRRK2 leads to co-ordinated responses in protein translation and trafficking and argue against a dominant negative role for the G2019S mutation.

Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation.

BACKGROUND: Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. METHODS: Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. RESULTS: Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. CONCLUSIONS: Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.