Mitochondrial Dysfunction and Decrease in Body Weight of a Transgenic Knock-in Mouse Model for TDP-43

The majority of amyotrophic lateral sclerosis (ALS) cases as well as many patients suffering from frontotemporal lobar dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR DNA-binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes, whereas the endogenous mouse Tdp-43 was decreased to 20% of wild type levels as a result of disturbed feedback regulation. Heterozygous TDP-43^A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals, we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol, and glucose in the blood. As seen in transmission electron microscopy, neuronal cells in motor cortices of TDP-43^A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90%, but only slight motoric impairment was detected. The observed phenotype was interpreted as a predisease model, which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.     

Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes: IMPLICATIONS FOR DISEASE SPREADING AND MOTOR NEURON PATHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS*

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.     

Intranuclear Aggregation of Mutant FUS/TLS as a Molecular Pathomechanism of Amyotrophic Lateral Sclerosis

Dominant mutations in FUS/TLS cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for aggregation of FUS in vitro and in vivo. Spontaneous in vitro formation of amyloid-like fibrillar aggregates was observed in mutant but not wild-type FUS, and notably, those fibrils functioned as efficient seeds to trigger the aggregation of wild-type protein. In addition, the G156E mutation did not disturb the nuclear localization of FUS but facilitated the formation of intranuclear inclusions in rat hippocampal neurons with significant cytotoxicity. We thus propose that intranuclear aggregation of FUS triggered by a subset of pathogenic mutations is an alternative pathomechanism of FUS-related fALS diseases.     

DJ-1 Is a Copper Chaperone Acting on SOD1 Activation

Lack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested based on a copper-mediated interaction between the two proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions. We structurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis.     

Neuronal Exosomal miRNA-dependent Translational Regulation of Astroglial Glutamate Transporter GLT1

Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.     

Disulfide Scrambling Describes the Oligomer Formation of Superoxide Dismutase (SOD1) Proteins in the Familial Form of Amyotrophic Lateral Sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) are a cause of a familial form of amyotrophic lateral sclerosis. Wild-type SOD1 forms a highly conserved intra-molecular disulfide bond, whereas pathological SOD1 proteins are cross-linked via intermolecular disulfide bonds and form insoluble oligomers. A thiol-disulfide status in SOD1 will thus play a regulatory role in determining its folding/misfolding pathways; however, it remains unknown how pathogenic mutations in SOD1 affect the thiol-disulfide status to facilitate the protein misfolding. Here, we show that the structural destabilization of SOD1 scrambles a disulfide bond among four Cys residues in an SOD1 molecule. The disulfide scrambling produces SOD1 monomers with distinct electrophoretic mobility and also reproduces the formation of disulfide-linked oligomers. We have also found that the familial form of amyotrophic lateral sclerosis-causing mutations facilitate the disulfide scrambling in SOD1. Based upon our results, therefore, scrambling of the conserved disulfide bond will be a key event to cause the pathological changes in disease-associated mutant SOD1 proteins.     

Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.     

Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic amyotrophic lateral sclerosis (ALS) model mice

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.     

Misfolded Polyglutamine,Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively “misfolded” state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.     

Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice

Mutations in the metalloenzyme copper-zinc superoxide dismutase (SOD1) cause one form of familial amyotrophic lateral sclerosis (ALS), and metals are suspected to play a pivotal role in ALS pathology. To learn more about metals in ALS, we determined the metallation states of human wild-type or mutant (G37R, G93A, and H46R/H48Q) SOD1 proteins from SOD1-ALS transgenic mice spinal cords. SOD1 was gently extracted from spinal cord and separated into insoluble (aggregated) and soluble (supernatant) fractions, and then metallation states were determined by HPLC inductively coupled plasma MS. Insoluble SOD1-rich fractions were not enriched in copper and zinc. However, the soluble mutant and WT SOD1s were highly metallated except for the metal-binding-region mutant H46R/H48Q, which did not bind any copper. Due to the stability conferred by high metallation of G37R and G93A, it is unlikely that these soluble SOD1s are prone to aggregation in vivo, supporting the hypothesis that immature nascent SOD1 is the substrate for aggregation. We also investigated the effect of SOD1 overexpression and disease on metal homeostasis in spinal cord cross-sections of SOD1-ALS mice using synchrotron-based x-ray fluorescence microscopy. In each mouse genotype, except for the H46R/H48Q mouse, we found a redistribution of copper between gray and white matters correlated to areas of high SOD1. Interestingly, a disease-specific increase of zinc was observed in the white matter for all mutant SOD1 mice. Together these data provide a picture of copper and zinc in the cell as well as highlight the importance of these metals in understanding SOD1-ALS pathology.     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.     

Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.     

Folding of the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State

Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that is not observed in RRM1. Strikingly, a portion of RRM2 beginning at position 208, which mimics a cleavage site observed in patient tissues, increases the population of this intermediate state. Mutually stabilizing interactions between the domains in the tethered RRM1 and RRM2 construct reduce the population of the intermediate state and enhance DNA/RNA binding. Despite the high sequence homology of the two domains, a network of large hydrophobic residues in RRM2 provides a possible explanation for the increased stability of RRM2 compared with RRM1. The cluster analysis suggests that the intermediate state may play a functional role by enhancing access to the nuclear export signal contained within its sequence. The intermediate state may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation that may contribute to disease.     

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid: A LINK TO TOXIC EFFECTS OF CALCIUM OVERLOAD IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)?*

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca²⁺ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca²⁺. We show that at physiological pH, Ca²⁺ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca²⁺ boosts the onset of SOD1 aggregation. In agreement, Ca²⁺ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca²⁺ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca²⁺-induced aggregates, thus indicating that Ca²⁺ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca²⁺ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca²⁺ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.     

Structural Transformation of the Amyloidogenic Core Region of TDP-43 Protein Initiates Its Aggregation and Cytoplasmic Inclusion

TDP-43 (TAR DNA-binding protein of 43 kDa) is a major deposited protein in amyotrophic lateral sclerosis and frontotemporal dementia with ubiquitin. A great number of genetic mutations identified in the flexible C-terminal region are associated with disease pathologies. We investigated the molecular determinants of TDP-43 aggregation and its underlying mechanisms. We identified a hydrophobic patch (residues 318–343) as the amyloidogenic core essential for TDP-43 aggregation. Biophysical studies demonstrated that the homologous peptide formed a helix-turn-helix structure in solution, whereas it underwent structural transformation from an α-helix to a β-sheet during aggregation. Mutation or deletion of this core region significantly reduced the aggregation and cytoplasmic inclusions of full-length TDP-43 (or TDP-35 fragment) in cells. Thus, structural transformation of the amyloidogenic core initiates the aggregation and cytoplasmic inclusion formation of TDP-43. This particular core region provides a potential therapeutic target to design small-molecule compounds for mitigating TDP-43 proteinopathies.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.