Evidence of synergism among three genetic variants in a patient with LMNA-related lipodystrophy and amyotrophic lateral sclerosis leading to a remarkable nuclear phenotype.

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), can be clinically heterogeneous which may be explained by the co-inheritance of multiple genetic variants that modify the clinical course. In this study we examine variants in three genes in a family with one individual presenting with ALS and lipodystrophy. Sequencing revealed a p.Gly602Ser variant in LMNA, and two additional variants, one each in SETX (g.intron10-13delCTT) and FUS (p.Gly167_Gly168del). These latter genes have been linked to ALS. All family members were genotyped and each variant, and each combination of variants detected, were functionally evaluated in vitro regarding effects on cell survival, expression patterns and cellular phenotype. Muscle biopsy retrieved from the individual with ALS showed leakage of chromatin from the nucleus, a phenotype that was recapitulated in vitro with expression of all three variants simultaneously. Individually expressed variants gave cellular phenotypes there were unremarkable. Interestingly the FUS variant appears to be protective against the effects of the SETX and the LMNA variants on cell viability and may indicate loss of interaction of FUS with SETX and/or R-loops. We conclude that these findings support genetic modifications as an explanation of the clinical heterogeneity observed in human disease.

Tau protein phosphorylation at Thr175 initiates fibril formation via accessibility of the N-terminal phosphatase-activating domain.

One of the neuropathological hallmarks of the tauopathies is the formation of neuronal cytoplasmic inclusions and fibrils of microtubule-associated tau protein (tau). The phosphorylation of Thr175 of tau (pThr175 tau) appears to be sufficient for fibril formation in vitro and in vivo, but the mechanism by which this initiates fibril formation is unknown. Using transient transfections of tau mutants into HEK293T cells, we determined that the phosphorylation of Thr175 leads to exposure of the tau N-terminal phosphatase-activating domain (PAD). The exposed PAD is known to interact with protein phosphatase-1 (PP1) resulting in glycogen synthase kinase 3ß (GSK3ß) activation. In vivo, a single traumatic controlled cortical injury in rats also resulted in the phosphorylation of Thr175 and increased exposure of tau PAD followed by pathological tau fibril formation. Taken together, these data suggest that neurotoxicity may be precipitated by phosphorylation at Thr175 and subsequent tau PAD exposure, GSK3ß activation and tau fibril formation. Cover Image for this issue: doi: 10.1111/jnc.14767.

Inclusion Formation and Toxicity of the ALS Protein RGNEF and Its Association with the Microtubule Network.

The Rho guanine nucleotide exchange factor (RGNEF) protein encoded by the ARHGEF28 gene has been implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Biochemical and pathological studies have shown that RGNEF is a component of the hallmark neuronal cytoplasmic inclusions in ALS-affected neurons. Additionally, a heterozygous mutation in ARHGEF28 has been identified in a number of familial ALS (fALS) cases that may give rise to one of two truncated variants of the protein. Little is known about the normal biological function of RGNEF or how it contributes to ALS pathogenesis. To further explore RGNEF biology we have established and characterized a yeast model and characterized RGNEF expression in several mammalian cell lines. We demonstrate that RGNEF is toxic when overexpressed and forms inclusions. We also found that the fALS-associated mutation in ARGHEF28 gives rise to an inclusion-forming and toxic protein. Additionally, through unbiased screening using the split-ubiquitin system, we have identified RGNEF-interacting proteins, including two ALS-associated proteins. Functional characterization of other RGNEF interactors identified in our screen suggest that RGNEF functions as a microtubule regulator. Our findings indicate that RGNEF misfolding and toxicity may cause impairment of the microtubule network and contribute to ALS pathogenesis.

TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis.

See Fratta and Isaacs (doi:10.1093/brain/awy091) for a scientific commentary on this article.The RNA binding proteins TDP-43 (encoded by TARDBP) and hnRNP A1 (HNRNPA1) are each mutated in certain amyotrophic lateral sclerosis cases and are often mislocalized in cytoplasmic aggregates within motor neurons of affected patients. Cytoplasmic inclusions of TDP-43, which are accompanied by a depletion of nuclear TDP-43, are observed in most amyotrophic lateral sclerosis cases and nearly half of frontotemporal dementia cases. Here, we report that TDP-43 binds HNRNPA1 pre-mRNA and modulates its splicing, and that depletion of nuclear TDP-43 results in increased inclusion of a cassette exon in the HNRNPA1 transcript, and consequently elevated protein levels of an isoform containing an elongated prion-like domain, referred to as hnRNP A1B. Combined in vivo and in vitro approaches demonstrated greater fibrillization propensity for hnRNP A1B, which drives protein aggregation and is toxic to cells. Moreover, amyotrophic lateral sclerosis patients with documented TDP-43 pathology showed neuronal hnRNP A1B cytoplasmic accumulation, indicating that TDP-43 mislocalization may contribute to neuronal vulnerability and loss via altered HNRNPA1 pre-mRNA splicing and function. Given that TDP-43 and hnRNP A1 each bind, and thus modulate, a third of the transcriptome, our data suggest a much broader disruption in RNA metabolism than previously considered.

RNA and Protein Interactors with TDP-43 in Human Spinal-Cord Lysates in Amyotrophic Lateral Sclerosis.

The TAR DNA-binding protein of 43 kDa (TDP-43) is a dual function RNA- and DNA-binding protein with varied cellular functions. In degenerating motor neurons in amyotrophic lateral sclerosis (ALS), TDP-43 relocalizes from the nucleus to the cytosol, where it is sequestered into inclusions. It is likely that the pathogenic role of TDP-43 in ALS can involve either a gain or a loss of function, depending on the nature of its RNA or protein interactor. However, while TDP-43 binding partners have been identified in a range of model systems and from the human brain, interactors from human spinal-cord tissue have not. In this study, we have characterized both protein and RNA TDP-43 interactors from neuropathologically normal (control) and ALS-affected ventral lumbar spinal cord, including sporadic ALS (sALS) and familial cases harboring either a A4T mutant SOD1 or a 3' UTR *c.41G>A mutant FUS/TLS or expressing pathological c9orf72 expanded repeats. RNA interactors with TDP-43 were similar between the control and ALS spinal cords examined regardless of genotype. In contrast, protein interactors with TDP-43 did demonstrate differences, with the sALS and mtSOD1 harboring cases examined differing from the protein interactors identified in the FUS 3' UTR mutation and c9orf72 repeat-positive cases.

Rho guanine nucleotide exchange factor (RGNEF) is a prosurvival factor under stress conditions.

Rho guanine nucleotide exchange factor (RGNEF) is a 190kDa RNA binding protein (RBP) that also contains a Dbl/PH domain capable of RhoA activation. Consistent with a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS), RGNEF forms pathological neuronal cytoplasmic inclusions in degenerating spinal motor neurons. To further understand the role of RGNEF in the stress response, we first observed that the expression of RGNEF is upregulated in murine spinal motor neurons following distal sciatic nerve injury. Secondly, in response to in vitro cellular stress (500µM sodium arsenite for 1h; or 400mM sorbitol 1 hour exposure; as an oxidative or osmotic stress, respectively), we observed a significant survival benefit in RGNEF-transfected HEK293T cells. Using deletion constructs, we found that the NH2-terminus domain is essential for this protective effect. Interestingly, we observed that under stress conditions RGNEF associates with Staufen1 positive granules but not TIA-1-positive stress granules. These findings support the hypothesis that RGNEF plays a critical role both in RNA homeostasis and in the response to cell stress.

RNA-binding proteins as molecular links between cancer and neurodegeneration.

For many years, epidemiological studies have suggested an association between cancer and neurodegenerative disorders-two disease processes that seemingly have little in common. Although these two disease processes share disruptions in a wide range of cellular pathways, including cell survival, cell death and the cell cycle, the end result is very divergent: uncontrolled cell survival and proliferation in cancer and progressive neuronal cell death in neurodegeneration. Despite the clinical data connecting these two disease processes, little is known about the molecular links between them. Among the mechanisms affected in cancer and neurodegenerative diseases, alterations in RNA metabolism are obtaining significant attention given the critical role for RNA transcription, maturation, transport, stability, degradation and translation in normal cellular function. RNA-binding proteins (RBPs) are integral to each stage of RNA metabolism through their participation in the formation of ribonucleoprotein complexes (RNPs). RBPs have a broad range of functions including posttranscriptional regulation of mRNA stability, splicing, editing and translation, mRNA export and localization, mRNA polyadenylation and miRNA biogenesis, ultimately impacting the expression of every single gene in the cell. In this review, we examine the evidence for RBPs as being key a molecular linkages between cancer and neurodegeneration.

Comprehensive luciferase-based reporter gene assay reveals previously masked up-regulatory effects of miRNAs.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the majority of the transcriptome at a post-transcriptional level. Because of this critical role, it is important to ensure that the assays used to determine their functionality are robust and reproducible. Typically, the reporter gene assay in cell-based systems has been the first-line method to study miRNA functionality. In order to overcome some of the potential errors in interpretation that can be associated with this assay, we have developed a detailed protocol for the luciferase reporter gene assay that has been modified for miRNAs. We demonstrate that normalization against the effect of the miRNA and cellular factors on the luciferase coding sequence is essential to obtain the specific impact of the miRNA on the 3'UTR (untranslated region) target. Our findings suggest that there is a real possibility that the roles for miRNA in transcriptome regulation may be misreported due to inaccurate normalization of experimental data and also that up-regulatory effects of miRNAs are not uncommon in cells. We propose to establish this comprehensive method as standard for miRNA luciferase reporter assays to avoid errors and misinterpretations in the functionality of miRNAs.

New metal-organic framework coated sodium alginate for the delivery of curcumin as a sustainable drug delivery and cancer therapy system.

The utilization of biocompatible drug delivery systems with extended drug release capabilities is highly advantageous in cancer therapy, as they can mitigate adverse effects. To establish such a biocompatible system with prolonged drug release behavior, researchers developed an innovative drug carrier. In this study, a sustainable approach was employed to synthesize a new zinc-based metal-organic framework (Zn-MOF) through the reaction between synthesized Schiff base ligands and zinc ions. Comprehensive analyses, including FT-IR, XRD, SEM, BET surface area, and TGA techniques, were employed to thoroughly characterize the frameworks. Following comprehensive characterization, curcumin (CUR) was loaded onto the Zn-MOF, resulting in CUR entrapment efficiency and loading capacity of 79.23 % and 26.11 %, respectively. In vitro evaluations of CUR release from CUR@MOF exhibited controlled release patterns, releasing 78.9 % and 50.0 % of CUR at pH 5.0 and pH 7.4, respectively. To mitigate initial burst release, a coating of the biopolymer sodium alginate (SA) was applied to CUR@Zn-MOF. In vitro CUR release tests indicated that SA/CUR@Zn-MOF outperformed pristine CUR@Zn-MOF. The release of CUR conformed to the Korsmeyer-Peppas model, displaying non-Fickian diffusion. Furthermore, an in vitro cytotoxicity study clearly demonstrated the potent anti-tumor activity of the synthesized CUR@Zn-MOF attributed to its controlled release of CUR. This led to the induction of apoptotic effects and cell death across HeLa, HEK293, and SH-SY5Y cell lines. These findings strongly suggest that the developed pH-sensitive carriers hold remarkable potential as targeted vehicles for drug delivery in cancer therapy.

A technique for repeated blood and cerebrospinal fluid sampling from individual rats over time without the need for repeated anesthesia.

Ethical animal use follows the 3R's: Replacement, Reduction and Refinement. Here, we present the use of simultaneous jugular vein and cisterna magna catheterization via a port system in rats for repeated fluid sampling for 14 consecutive days without loss of catheter patency. This technique allows repeated intra-animal sampling without anesthesia and, if used with pooling samples from a cohort of animals, replaces the need for terminal collections for sufficient sample volumes.

A methodological primer of extracellular vesicles isolation and characterization via different techniques.

We present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at -80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the "best method" for their work under their individual conditions.

Ataxin-2 repeat-length variation and neurodegeneration.

Expanded glutamine repeats of the ataxin-2 (ATXN2) protein cause spinocerebellar ataxia type 2 (SCA2), a rare neurodegenerative disorder. More recent studies have suggested that expanded ATXN2 repeats are a genetic risk factor for amyotrophic lateral sclerosis (ALS) via an RNA-dependent interaction with TDP-43. Given the phenotypic diversity observed in SCA2 patients, we set out to determine the polymorphic nature of the ATXN2 repeat length across a spectrum of neurodegenerative disorders. In this study, we genotyped the ATXN2 repeat in 3919 neurodegenerative disease patients and 4877 healthy controls and performed logistic regression analysis to determine the association of repeat length with the risk of disease. We confirmed the presence of a significantly higher number of expanded ATXN2 repeat carriers in ALS patients compared with healthy controls (OR = 5.57; P= 0.001; repeat length >30 units). Furthermore, we observed significant association of expanded ATXN2 repeats with the development of progressive supranuclear palsy (OR = 5.83; P= 0.004; repeat length >30 units). Although expanded repeat carriers were also identified in frontotemporal lobar degeneration, Alzheimer's and Parkinson's disease patients, these were not significantly more frequent than in controls. Of note, our study identified a number of healthy control individuals who harbor expanded repeat alleles (31-33 units), which suggests caution should be taken when attributing specific disease phenotypes to these repeat lengths. In conclusion, our findings confirm the role of ATXN2 as an important risk factor for ALS and support the hypothesis that expanded ATXN2 repeats may predispose to other neurodegenerative diseases, including progressive supranuclear palsy.

Upregulation of LRRK2 following traumatic brain injury does not directly phosphorylate Thr175 tau.

Phosphorylated microtubule-associated protein tau (tau) aggregates are a pathological hallmark of various neurodegenerative diseases, including chronic traumatic encephalopathy and amyotrophic lateral sclerosis with cognitive impairment. While there are many residues phosphorylated on tau, phosphorylation of threonine 175 (pThr175 tau) has been shown to initiate fibril formation in vitro and is present in pathological tau aggregates in vivo. Given this, preventing Thr175 tau phosphorylation presents a potential approach to reduce fibril formation; however, the kinase(s) acting on Thr175 are not yet fully defined. Using a single controlled cortical impact rodent model of traumatic brain injury (TBI), which rapidly induces Thr175 tau phosphorylation, we observed an upregulation and alteration in subcellular localization of leucine-rich repeat kinase 2 (LRRK2), a kinase that has been implicated in tau phosphorylation. LRRK2 upregulation was evident by one-day post-injury and persisted to day 10. The most notable changes were observed in microglia at the site of injury in the cortex. To determine if the appearance of pThr175 tau was causally related to the upregulation of LRRK2 expression, we examined the ability of LRRK2 to phosphorylate Thr175in vitro by co-transfecting 2N4R human WT-tau with either LRRK2-WT, constitutively-active LRRK2-G2019S or inactive LRRK2-3XKD. We found no significant difference in the level of pThr175 tau between the overexpression of LRRK2-WT, -G2019S or -3XKD, suggesting LRRK2 does not phosphorylate tau at Thr175. Further, downstream events known to follow Thr175 phosphorylation and known to be associated with pathological tau fibril formation (pSer9-GSK3ß and pThr231 tau induction) also remained unchanged. We conclude that while LRRK2 expression is altered in TBI, it does not contribute directly to pThr175 tau generation.

Co-aggregation of RNA binding proteins in ALS spinal motor neurons: evidence of a common pathogenic mechanism.

While the pathogenesis of amyotrophic lateral sclerosis (ALS) remains to be clearly delineated, there is mounting evidence that altered RNA metabolism is a commonality amongst several of the known genetic variants of the disease. In this study, we evaluated the expression of 10 ALS-associated proteins in spinal motor neurons (MNs) in ALS patients with mutations in C9orf72 (C9orf72(GGGGCC)-ALS; n = 5), SOD1 (mtSOD1-ALS; n = 9), FUS/TLS (mtFUS/TLS-ALS; n = 2), or TARDBP (mtTDP-43-ALS; n = 2) and contrasted these to cases of sporadic ALS (sALS; n = 4) and familial ALS without known mutations (fALS; n = 2). We performed colorimetric immunohistochemistry (IHC) using antibodies against TDP-43, FUS/TLS, SOD1, C9orf72, ubiquitin, sequestosome 1 (p62), optineurin, phosphorylated high molecular weight neurofilament, peripherin, and Rho-guanine nucleotide exchange factor (RGNEF). We observed that RGNEF-immunoreactive neuronal cytoplasmic inclusions (NCIs) can co-localize with TDP-43, FUS/TLS and p62 within spinal MNs. We confirmed their capacity to interact by co-immunoprecipitations. We also found that mtSOD1-ALS cases possess a unique IHC signature, including the presence of C9orf72-immunoreactive diffuse NCIs, which allows them to be distinguished from other variants of ALS at the level of light microscopy. These findings support the hypothesis that alterations in RNA metabolism are a core pathogenic pathway in ALS. We also conclude that routine IHC-based analysis of spinal MNs may aid in the identification of families not previously suspected to harbor SOD1 mutations.

Human low molecular weight neurofilament (NFL) mRNA interacts with a predicted p190RhoGEF homologue (RGNEF) in humans.

In the mouse, p190RhoGEF is a low molecular weight neurofilament (NFL) mRNA stability factor that is involved in NF aggregate formation in neurons. A human homologue of this protein has not been described. Our objective was to identify a human homologue of p190RhoGEF, and to determine its interaction with human NFL mRNA. We used sequence homology searches to predict a human homologue (RGNEF), and RT-PCR to determine the expression of mRNA in ALS and neuropathologically normal control tissues. Gel shift assays determined the interaction of RGNEF with human NFL mRNA in vitro, while IP-RT-PCR and gel shift assays were used to confirm the interaction in tissue lysates. We determined that RGNEF is a human homologue of p190RhoGEF, and that its RNA is expressed in both brain and spinal cord. While RGNEF and NFL mRNA interact directly in vitro, interestingly they only appear to interact in ALS lysates and not in controls. These data add another player to the family of NFL mRNA stability regulators, and raise the intriguing possibility that the mechanism by which p190RhoGEF contributes to murine neuronal NF aggregate formation may be important to human ALS NF aggregate formation.

Neurofilament Immunohistochemistry Followed by Luxol Fast Blue, for Staining Axons and Myelin in the Same Paraffin Section of Spinal Cord.

Many disorders of the central nervous system are characterized by both axonal pathology and demyelination. In assessing this concurrent pathology, techniques for staining axons or myelin are frequently used separately. Here we report the development of a combined immunohistochemical and tinctorial staining technique in which we have modified the Luxol fast blue myelin stain to be used in conjunction with a diaminobenzidine-based immunohistochemical stain for high molecular weight neurofilament (SMI-31). This modification of staining will have utility in experimental neuropathology laboratories investigating demyelination and axonal damage in human tissue and animal models.

Alterations in Tau Metabolism in ALS and ALS-FTSD.

There is increasing acceptance that amyotrophic lateral sclerosis (ALS), classically considered a neurodegenerative disease affecting almost exclusively motor neurons, is syndromic with both clinical and biological heterogeneity. This is most evident in its association with a broad range of neuropsychological, behavioral, speech and language deficits [collectively termed ALS frontotemporal spectrum disorder (ALS-FTSD)]. Although the most consistent pathology of ALS and ALS-FTSD is a disturbance in TAR DNA binding protein 43 kDa (TDP-43) metabolism, alterations in microtubule-associated tau protein (tau) metabolism can also be observed in ALS-FTSD, most prominently as pathological phosphorylation at Thr175 (pThr175tau). pThr175 has been shown to promote exposure of the phosphatase activating domain (PAD) in the tau N-terminus with the consequent activation of GSK3ß mediated phosphorylation at Thr231 (pThr231tau) leading to pathological oligomer formation. This pathological cascade of tau phosphorylation has been observed in chronic traumatic encephalopathy with ALS (CTE-ALS) and in both in vivo and in vitro experimental paradigms, suggesting that it is of critical relevance to the pathobiology of ALS-FTSD. It is also evident that the co-existence of alterations in the metabolism of TDP-43 and tau acts synergistically in a rodent model to exacerbate the pathology of either.

Tau phosphorylation at threonine-175 leads to fibril formation and enhanced cell death: implications for amyotrophic lateral sclerosis with cognitive impairment.

Although amyotrophic lateral sclerosis (ALS) can be associated with cognitive impairment (ALSci) as a reflection of frontotemporal lobar degeneration, the basis of this process is unknown. The observation of neuronal and extraneuronal tau deposition in ALSci in addition to a unique tau phosphorylation at Thr175 has suggested that ALSci can be associated with alterations in tau metabolism. We have examined the association between phosphorylation at Thr175 and tau fibril formation. Both soluble and insoluble tau was purified from control, patients with Alzheimer's disease (AD), ALS without cognitive impairment, and ALSci and the tendency to fibril formation assayed ex vivo using the thioflavin S fluorescence assay. The extent of fibril formation was significantly greater in tau derived from ALSci, with ALS-derived tau being intermediate between control and AD-derived tau. Using both Neuro2A and human embryonic kidney (HEK293T) cells, we expressed full-length tau constructs harboring either a pseudophosphorylation at Thr175 (Thr175-Asp-tau), inhibition of Thr175 phosphorylation (Thr175-Ala-tau) or intact tau (wild-type tau). Both tau fibril formation and cell death were significantly enhanced in the presence of Thr175-Asp-tau, regardless of the tau isoform, suggesting that phosphorylation of Thr175 is associated with tau fibril formation in ALSci.