Proteomic analysis of X-linked dystonia parkinsonism disease striatal neurons reveals altered RNA metabolism and splicing.

X-linked dystonia-parkinsonism (XDP) is a rare neurodegenerative disease endemic to the Philippines. The genetic cause for XDP is an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within intron 32 of TATA-binding protein associated factor 1 (TAF1) that causes an alteration of TAF1 splicing, partial intron retention, and decreased transcription. Although TAF1 is expressed in all organs, medium spiny neurons (MSNs) within the striatum are one of the cell types most affected in XDP. To define how mutations in the TAF1 gene lead to MSN vulnerability, we carried out a proteomic analysis of human XDP patient-derived neural stem cells (NSCs) and MSNs derived from induced pluripotent stem cells. NSCs and MSNs were grown in parallel and subjected to quantitative proteomic analysis in data-independent acquisition mode on the Orbitrap Eclipse Tribrid mass spectrometer. Subsequent functional enrichment analysis demonstrated that neurodegenerative disease-related pathways, such as Huntington's disease, spinocerebellar ataxia, cellular senescence, mitochondrial function and RNA binding metabolism, were highly represented. We used weighted coexpression network analysis (WGCNA) of the NSC and MSN proteomic data set to uncover disease-driving network modules. Three of the modules significantly correlated with XDP genotype when compared to the non-affected control and were enriched for DNA helicase and nuclear chromatin assembly, mitochondrial disassembly, RNA location and mRNA processing. Consistent with aberrant mRNA processing, we found splicing and intron retention of TAF1 intron 32 in XDP MSN. We also identified TAF1 as one of the top enriched transcription factors, along with YY1, ATF2, USF1 and MYC. Notably, YY1 has been implicated in genetic forms of dystonia. Overall, our proteomic data set constitutes a valuable resource to understand mechanisms relevant to TAF1 dysregulation and to identify new therapeutic targets for XDP.

Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for development of neuroprotective strategies for PD.

Neuroinflammation and α-synuclein accumulation in response to glucocerebrosidase deficiency are accompanied by synaptic dysfunction.

Clinical, epidemiological and experimental studies confirm a connection between the common degenerative movement disorder Parkinson's disease (PD) that affects over 1 million individuals, and Gaucher disease, the most prevalent lysosomal storage disorder. Recently, human imaging studies have implicated impaired striatal dopaminergic neurotransmission in early PD pathogenesis in the context of Gaucher disease mutations, but the underlying mechanisms have yet to be characterized. In this report we describe and characterize two novel long-lived transgenic mouse models of Gba deficiency, along with a subchronic conduritol-ß-epoxide (CBE) exposure paradigm. All three murine models revealed striking glial activation within nigrostriatal pathways, accompanied by abnormal α-synuclein accumulation. Importantly, the CBE-induced, pharmacological Gaucher mouse model replicated this change in dopamine neurotransmission, revealing a markedly reduced evoked striatal dopamine release (approximately 2-fold) that indicates synaptic dysfunction. Other changes in synaptic plasticity markers, including microRNA profile and a 24.9% reduction in post-synaptic density size, were concomitant with diminished evoked dopamine release following CBE exposure. These studies afford new insights into the mechanisms underlying the Parkinson's-Gaucher disease connection, and into the physiological impact of related abnormal α-synuclein accumulation and neuroinflammation on nigrostriatal dopaminergic neurotransmission.

LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.

Parkinson's disease associated mutations in leucine rich repeat kinase 2 (LRRK2) impair mitochondrial function and increase the vulnerability of induced pluripotent stem cell (iPSC)-derived neural cells from patients to oxidative stress. Since mitochondrial DNA (mtDNA) damage can compromise mitochondrial function, we examined whether LRRK2 mutations can induce damage to the mitochondrial genome. We found greater levels of mtDNA damage in iPSC-derived neural cells from patients carrying homozygous or heterozygous LRRK2 G2019S mutations, or at-risk individuals carrying the heterozygous LRRK2 R1441C mutation, than in cells from unrelated healthy subjects who do not carry LRRK2 mutations. After zinc finger nuclease-mediated repair of the LRRK2 G2019S mutation in iPSCs, mtDNA damage was no longer detected in differentiated neuroprogenitor and neural cells. Our results unambiguously link LRRK2 mutations to mtDNA damage and validate a new cellular phenotype that can be used for examining pathogenic mechanisms and screening therapeutic strategies.

Effects of cadmium chloride on mouse inner medullary collecting duct cells.

Cadmium is a known renal toxin. The cytotoxic effect of cadmium chloride (CdCl2) was evaluated on renal inner medullary collecting duct cells (mIMCD3). The 24 hr LC50 value for CdCl2 in mIMCD3 cells was 40 µM. The present study showed that mIMCD3 cells were sensitive to CdCl2 exposure.

Small molecules greatly improve conversion of human-induced pluripotent stem cells to the neuronal lineage.

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.

Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.

Mitochondrial dysfunction has been frequently implicated in the neurodegenerative process that underlies Parkinson's disease (PD), but the basis for this impairment is not fully understood. The goal of this study was to investigate the effects of α-synuclein (α-syn) gene multiplication on mitochondrial function in human tissue. To investigate this question, human fibroblasts were taken from a patient with parkinsonism carrying a triplication in the α-syn gene. Unexpectedly, the cells showed a significant decrease in cell growth compared to matched healthy controls. With regard to mitochondrial function, α-syn triplication fibroblasts exhibited a 39% decrease in ATP production, a 40% reduction in mitochondrial membrane potential, and a 49% reduction in complex I activity. Furthermore, they proved to be more sensitive to the effects of the nigrostrial toxicant paraquat compared to controls. Finally, siRNA knockdown of α-syn resulted in a partial rescue of mitochondrial impairment and reduction of paraquat-induced cell toxicity, suggesting that α-syn plays a causative role for mitochondrial dysfunction in these patient-derived peripheral skin fibroblasts.

Alpha-synuclein suppression by targeted small interfering RNA in the primate substantia nigra.

The protein alpha-synuclein is involved in the pathogenesis of Parkinson's disease and other neurodegenerative disorders. Its toxic potential appears to be enhanced by increased protein expression, providing a compelling rationale for therapeutic strategies aimed at reducing neuronal alpha-synuclein burden. Here, feasibility and safety of alpha-synuclein suppression were evaluated by treating monkeys with small interfering RNA (siRNA) directed against alpha-synuclein. The siRNA molecule was chemically modified to prevent degradation by exo- and endonucleases and directly infused into the left substantia nigra. Results compared levels of alpha-synuclein mRNA and protein in the infused (left) vs. untreated (right) hemisphere and revealed a significant 40-50% suppression of alpha-synuclein expression. These findings could not be attributable to non-specific effects of siRNA infusion since treatment of a separate set of animals with luciferase-targeting siRNA produced no changes in alpha-synuclein. Infusion with alpha-synuclein siRNA, while lowering alpha-synuclein expression, had no overt adverse consequences. In particular, it did not cause tissue inflammation and did not change (i) the number and phenotype of nigral dopaminergic neurons, and (ii) the concentrations of striatal dopamine and its metabolites. The data represent the first evidence of successful anti-alpha-synuclein intervention in the primate substantia nigra and support further development of RNA interference-based therapeutics.

Lysosomal degradation of alpha-synuclein in vivo.

Pathologic accumulation of alpha-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of alpha-synuclein in vivo was indicated by the detection of alpha-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal alpha-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of alpha-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with alpha-synuclein within nigral dopaminergic neurons of paraquat-treated and alpha-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of alpha-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of alpha-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal alpha-synuclein buildup.

Decreased alpha-synuclein expression in the aging mouse substantia nigra.

Because of its normal function in synaptic plasticity and pathologic involvement in age-related neurodegenerative diseases, the protein alpha-synuclein could play an important role in aging processes. Here we compared alpha-synuclein expression in the substantia nigra and other brain regions of young (2-month-old), middle-aged (10-month-old), and old (20-month-old) mice. Levels of nigral alpha-synuclein mRNA, as assessed by both in situ hybridization and qPCR, were high in young mice and progressively declined in middle-aged and old animals. This age-dependent mRNA loss was paralleled by a marked reduction of alpha-synuclein protein; immunoreactivity of midbrain sections stained with an anti-alpha-synuclein antibody was most robust in 2-month-old mice and weakest in 20-month-old animals. Lowering of nigral alpha-synuclein could not be explained by a loss of dopaminergic neurons and was relatively specific since no change in beta-synuclein mRNA and protein occurred with advancing age. Finally, age-related decreases in alpha-synuclein were widespread throughout the mouse brain, affecting other regions (e.g., hippocampus) besides the substantia nigra. The data suggest that loss of alpha-synuclein could contribute to or be a marker of synaptic dysfunction in the aging brain. They also emphasize important differences in alpha-synuclein expression between rodents and primates since earlier reports have shown a marked elevation of alpha-synuclein protein in the substantia nigra of older humans and non-human primates.

Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys.

alpha-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic accumulation of alpha-synuclein and protein modifications (i.e. nitration, phosphorylation, and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of alpha-synuclein but not of beta-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy also revealed large numbers of dystrophic axons labeled with alpha-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) alpha-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. After toxicant exposure, alpha-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for alpha-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce alpha-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of alpha-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.

Determination of cytotoxicity of nephrotoxins on murine and human kidney cell lines.

The present study investigates renal inner medullary collecting duct (mIMCD3) cells and human embryonic kidney cells (HEK293) for evaluation of cytotoxicity of nephrotoxic compounds. The 24 h LC(50) values for cisplatin, paraquat and ibuprofen in mIMCD3 cells were 135, 155 and 3600 microM, respectively. The 24 h LC(50) values for paraquat and ibuprofen in HEK293 cells were 180 and 1000 microM, respectively. Effects of hyperosmolality on cytotoxicity of paraquat were additive in mIMCD3 cells. These data demonstrate that renal hyperosmolality has an additive effect on cytoxicity of paraquat.

Evaluation of cytotoxicity attributed to thimerosal on murine and human kidney cells.

Renal inner medullary collecting duct cells (mIMCD3) and human embryonic kidney cells (HEK293) were used for cytoscreening of thimerosal and mercury chloride (HgCl2). Thimerosal and HgCl2 acted in a concentration-dependent manner. In mIMCD3 cells the 24-h LC50 values for thimerosal, thiosalicylic acid, 2,2-dithiosalicylic acid, and 2-sulfobenzoic acid were 2.9, 2200, >1000, and >10,000 microM, respectively. The 24-h LC50 value for HgCl2 in mIMCD3 cells was 40 microM. In HEK293 cells, the 24-h LC50 value for thimerosal was 9.5 microM. These data demonstrate that the higher cytotoxicity produced by thimerosal on renal cells with respect to similar compounds without Hg may be related to this metal content. The present study also establishes mIMCD3 cells as a valuable model for evaluation of cytotoxicity of nephrotoxic compounds.

Specific TSC22 domain transcripts are hypertonically induced and alternatively spliced to protect mouse kidney cells during osmotic stress.

We recently cloned a novel osmotic stress transcription factor 1 (OSTF1) from gills of euryhaline tilapia (Oreochromis mossambicus) and demonstrated that acute hyperosmotic stress transiently increases OSTF1 mRNA and protein abundance [Fiol DF, Kültz D (2005) Proc Natl Acad Sci USA102, 927-932]. In this study, a genome-wide search was conducted to identify nine distinct mouse transforming growth factor (TGF)-beta-stimulated clone 22 domain (TSC22D) transcripts, including glucocorticoid-induced leucine zipper (GILZ), that are orthologs of OSTF1. These nine TSC22D transcripts are encoded at four loci on chromosomes 14 (TSC22D1, two splice variants), 3 (TSC22D2, four splice variants), X (TSC22D3, two splice variants), and 5 (TSC22D4). All nine mouse TSC22D transcripts are expressed in renal cortex, medulla and papilla, and in the mIMCD3 cell line. The two TSC22D3 transcripts (including GILZ) are upregulated by aldosterone but not by hyperosmolality in mIMCD3 cells. In contrast, TSC22D4 is stably upregulated by hyperosmolality in mIMCD3 cells and increased in renal papilla compared with cortex. Moreover, all four TSC22D2 transcripts are transiently upregulated by hyperosmolality and resemble tilapia OSTF1 in this regard. All TSC22D2 transcripts depend on hypertonicity as the signal for their upregulation and are unresponsive to increases in cell-permeable osmolytes. mRNA stabilization is the mechanism for TSC22D2 upregulation by hyperosmolality. Overexpression of TSC22D2-4 in mIMCD3 cells confers protection towards osmotic stress, as evidenced by a 2.7-fold increase in cell survival after 3 days at 600 mOsmol x kg(-1). Based on variable responsiveness to aldosterone and hyperosmolality in kidney cells we conclude that mouse TSC22D genes have diverse physiological functions. TSC22D2 and TSC22D4 are involved in adaptation of renal cells to hypertonicity suggesting that they represent important elements of osmosensory signal transduction in mouse kidney cells.

Nek8 mutation causes overexpression of galectin-1, sorcin, and vimentin and accumulation of the major urinary protein in renal cysts of jck mice.

The jck murine model, which results from a double point mutation in the nek8 gene, has been used to study the mechanism of autosomal recessive polycystic kidney disease (ARPKD). The renal proteome of jck mice was characterized by two-dimensional gel electrophoresis combined with mass spectrometry (MALDI-TOF/TOF). Four newly identified proteins were found to accumulate in the kidneys of jck mice with polycystic kidney disease (PKD) compared with their wild-type littermates. The proteins galectin-1, sorcin, and vimentin were found to be induced 9-, 9-, and 25-fold, respectively, in the PKD proteome relative to the wild type. The identity of these proteins was established by peptide mass fingerprinting and de novo MS/MS sequencing of selected peptides. Up-regulation of these three proteins may be due to the nek8 mutation, and their function may be related to the signaling and structural processes in the primary cilium. Additionally a series of protein isoforms observed only in the ARPKD kidney was identified as the major urinary protein (MUP). Peptide sequencing demonstrated that the isoforms MUP1, MUP2, and MUP6 are contained in this series. The MUP series showed a number of male-specific isoforms and a phosphorylation of the entire series with an increasing degree of phosphorylation of the acidic isoforms. In addition, the MUP series was localized to the cyst fluid of PKD mice, and a cellular mislocalization of galectin-1, sorcin, and vimentin in PKD tubular epithelial cells was shown. The abnormal and extremely high accumulation of the MUPs in the ARPKD kidney may be linked to a defect in protein transport and secretion. The discovery of these proteins will provide new information on the molecular and cellular processes associated with the mechanism of ARPKD.

Gadd45 proteins induce G2/M arrest and modulate apoptosis in kidney cells exposed to hyperosmotic stress.

Gadd45 proteins are induced by hyperosmolality in renal inner medullary (IM) cells, but their role for cell adaptation to osmotic stress is not known. We show that a cell line derived from murine renal IM cells responds to moderate hyperosmotic stress (540 mosmol/kg) by activation of G(2)/M arrest without significant apoptosis. If the severity of hyperosmotic stress exceeds the tolerance limit of this cell line (620 mosmol/kg) apoptosis is strongly induced. Using transient overexpression of ectopic Gadd45 proteins and simultaneous analysis of transfected versus non-transfected cells by laser-scanning cytometry, we were able to measure the effects of Gadd45 super-induction during hyperosmolality on G(2)/M arrest and apoptosis. Our results demonstrate that induction of all three Gadd45 isoforms inhibits mitosis and promotes G(2)/M arrest during moderate hyperosmotic stress but not in isosmotic controls. Furthermore, all three Gadd45 proteins are also involved in control of apoptosis during severe hyperosmotic stress. Under these conditions Gadd45gamma induction strongly potentiates apoptosis. In contrast, Gadd45alpha/beta induction transiently increases caspase 3/7 and annexin V binding before 12 h but inhibits later stages of apoptosis during severe hyperosmolality. These results show that Gadd45 isoforms function in common but also in distinct pathways during hyperosmolality and that their increased abundance contributes to the low mitotic index and protection of genomic integrity in cells of the mammalian renal inner medulla.