S-nitrosylated TDP-43 triggers aggregation,cell-to-cell spread,and neurotoxicity in hiPSCs and in vivo models of ALS/FTD

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of “sporadic” cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.

Identification of genetic, pathological, and clinical signatures of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) suggests a continuum of disease on a single ALS/FTD spectrum of disorders. At a genetic level, mutations in specific genes, including TARDBP (encoding the RNA-binding protein TDP-43), fused-in-sarcoma (FUS), and valosin containing protein (VCP), or hexanucleotide repeat expansion, as in C9ORF72, have been linked to ALS and/or FTD (1). Even in the absence of mutation, TDP-43 proteinopathy, involving cytoplasmic aggregation and consequent nuclear clearance of TDP-43 in affected cells, represents a major pathological hallmark appearing in 97% of ALS and 45% of FTD cases (2, 3). The high prevalence of TDP-43 proteinopathy in the face of rare genetic mutations in the TARDBP gene (representing only ∼4% of ALS cases) points to the possibility that other factors related to age or environment contribute to TDP-43 aggregation in the vast majority of patients with ALS/FTD spectrum disorders.Under normal conditions, TDP-43 resides predominantly in the nucleus, functioning as an RNA-binding protein for regulation of mRNA processing and stabilization (4, 5). In contrast, in ALS/FTD, TDP-43 becomes highly phosphorylated, ubiquitinated, and insoluble and mislocalizes to the cytosol to form stress granules (SGs) (6). For example, enhanced activity of casein kinases and possibly other kinases, such as glycogen synthase kinase 3 and cyclin-dependent kinases, lead to hyperphosphorylation of TDP-43, promoting aggregation of TDP-43 in cytosolic SGs (7, 8). SGs are nonmembrane-bound organelles that can sequester specific mRNAs via phase separation (9) to inhibit the initiation of translation. Additionally, emerging evidence suggests that cell-to-cell spreading of pathological aggregates of TDP-43 contributes to the propagation of the proteinopathy (10–12). Prior reports support the notion that disease progression in TDP-43 proteinopathy can be mediated by aggregation-related loss-of-normal function (e.g., RNA binding in the nucleus) or gain-of-toxic function, or possibly both (13). Studies have also implicated microglia-dependent pathways in TDP-43 proteinopathy (14). Furthermore, recent studies have revealed that TDP-43 pathology occurs in a wide variety of other neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) (15), suggesting a wider role for TDP-43 proteinopathy in neurodegeneration. However, mechanistic insight into the development and execution of TDP-43 pathology remains incompletely understood.Two risk factors that have been strongly implicated in the pathogenesis of neurodegenerative disorders are the aging process and environmental toxins (16–19). Both of these risk factors engender dramatic increases in chemically reactive species, including reactive oxygen and nitrogen species (ROS/RNS) such as nitric oxide (NO), and it has been suggested that this may play a role in the degenerative process as observed in ALS/FTD (18–22). Along these lines, we and others have demonstrated that protein S-nitrosylation, resulting from posttranslational modification of cysteine thiol groups by NO-related species, contributes to protein misfolding, mitochondrial dysfunction, synaptic impairment, and eventually neuronal cell loss (19). Several chemical mechanisms for in vivo formation of protein S-nitrosylation have been proposed (23, 24). For example, cysteine thiol (or more properly thiolate anion) can perform a reversible nucleophilic attack on a nitroso nitrogen to form a protein S-nitrosothiol via transnitrosation or transnitrosylation (24, 25). Mechanism notwithstanding, protein S-nitrosylation is now well recognized as a major contributor to both the physiological and pathophysiological activity (19, 20).Interestingly, the Food and Drug Administration (FDA)-approved drug edaravone (MCI-186) delays disease progression in some cases of ALS (26), possibly via scavenging RNS/ROS related to NO and hydroxyl radical groups (27, 28). While exogenous addition of ROS-generating agents has been reported to decrease the solubility of TDP-43 in vitro in cell-based models (29–31), endogenous reactive chemical species have not been previously reported to do this in a pathophysiologically relevant manner. Accordingly, in the present study, we report that not only exogenous but also endogenous RNS can trigger TDP-43 aggregation via S-nitrosylation and consequent disulfide bond formation; in models of FTD and ALS, we identify endogenous SNO-TDP-43 formation as a critical effector of pathological signaling, leading to its aggregation, altered RNA-binding activity, and neurotoxicity. Moreover, we find that increased expression of TDP-43 protein, as found in ALS, FTD, and other neurodegenerative disorders, results not only in misfolded/aggregated protein but also in dramatically increased NO production and additional protein misfolding/aggregation, representing a positive feed-forward loop to enhance nitrosative stress and thus protein misfolding. Additionally, we report that, in conjunction with NO, mutation in the VCP gene, as observed in some cases of ALS/FTD, triggers a dramatic increase in misfolded/aggregated TDP-43 in human-induced pluripotent stem cell (hiPSC)-derived motoneurons. Our results thus place TDP-43 at a unique node of intersection between genetic mutations associated with ALS/FTD and aging/environmental risk factors mediated by NO-related species, which together contribute to neurodegeneration in ALS/FTD spectrum disorders.     

Acid α-neuraminidase deficiency: a nephropathic phenotype?

Isolated neuraminidase deficiency is a member of the relatively rare group of storage disorders known as glycoproteinoses. We report the long-term natural history of the disease in one of the first patients described in the literature. An unusual feature of the disease is the abrupt onset and fulminant nature of the nephrotic syndrome, complications of which caused the demise of our patient. Pathological examination of the kidneys from this child revealed renal epithelial cell damage, most marked in the membranes of the glomeruli and proximal tubules, findings which are consistent with the high sialic acid content of the membrane in these areas of the nephron. Chemical analysis indicated that the bulk of the stored material in the kidney was in the form of polar sialyloligosaccharides of high molecular weight. On the basis of our experience, as well as the previous reports of neuraminidase-deficient patients with nephropathy, we propose a nephropathic phenotypic variant of type 2 infantile sialidosis.     

Epithelial cyst in cerebellopontine angle with xanthogranulomatous changes simulating cholesterol granuloma

We describe the clinical, radiological, and pathological features of an epithelial lined cyst in the cerebellopontine angle of a 54-year-old man who presented with headaches, ataxia, and multiple cranial nerve dysfunction. The surgically excised lesion showed a cyst lined by ciliated columnar epithelium with copious mucin secretion similar to that seen in colloid cysts of the third ventricle and enterogenous cysts of the spinal canal. In addition the cyst contained brownish material with an exuberant xanthogranulomatous reaction and numerous cholesterol clefts. This lesion closely resembled a cholesterol granuloma by radiographic and pathologic studies. Although two examples of neuroepithelial lined cysts have been described in the cerebellopontine angle, to our knowledge a lesion similar to that in our patient has not been reported previously.     

T-cell-receptor beta- and I-A beta-chain genes of normal SWR mice are linked with the development of lupus nephritis in NZB x SWR crosses.

The incidence of nephritis in autoimmune New Zealand Black (NZB) mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed the association of the I-A beta-chain gene of Ia-encoding region, the T-cell-receptor beta (TcR beta)-chain gene, and immunoglobulin heavy-chain allotype (IgH) with the development of lupus nephritis in 165 NZB X SWR crosses. We found that genes linked to the TcR and Ir gene loci of the normal SWR mice interacted with NZB-derived genes, leading to the development of accelerated and severe nephritis in the NZB X SWR crosses.     

Anti-inflammatory and irritant activities of curcumin analogues in rats

The anti-inflammatory activity of curcumin (C), sodium curcuminate (NaC), diacetyl curcumin (DAC), triethyl curcumin (TEC), tetrahydro curcumin (THC) and ferulic acid (FA) was compared with that of phenylbutazone (PB) using the carrageenin-induced rat paw edema and cotton pellet granuloma tests. The rank order of potencies of curcumin analogues and PB in carrageenininduced inflammation were NaC>THC>C>PB>TEC. The curcumin analogues decreased carrageenin-induced paw edema at low doses, however, at higher doses this effect was partially reversed. FA and DAC were devoid of antiinflammatory activity. Curcumin analogues were less effective in inhibiting the granulomatous tissue formation. Maximum activity was observed with TEC whereas C, NaC and PB were almost half as effective as TEC. C and NaC possess both anti-inflammatory and irritant properties as was evident from experiments in which drugs were incorporated into carrageenin and the cotton pellets for inducing inflammation. The anti-inflammatory action of NaC is not mediated through release of steroids from the adrenal cortex or inhibition of the biosynthesis of prostaglandins from arachidonic acid. The results of the present study support the rationale for the use of powdered rhizome of turmeric (contains 0.6% of curcumin) for conditions of sprain and inflammation.     

Characterization of lactate dehydrogenase of Plasmodium knowlesi

Some properties of purified lactate dehydrogenase, (EC. 1.1.1.27) from schizonts of Plasmodium knowlesi are described. The plasmodial enzyme migrated as single entity on polyacrylamide gel, and resembled rabbit muscle (M4) lactate dehydrogenase in its electrophoretic mobility. The P. knowlesi enzyme consisted of four identical subunits of 31 kDa. Purified lactate dehydrogenase was inhibited almost completely when incubated with 100 microM p-chloromercuribenzoate, Ag+ or Hg+ and such inhibition could be reversed by the addition of beta-mercaptoethanol or L-cysteine. Metal chelators did not show any remarkable effect. Oxalic acid is a competitive inhibitor of pyruvate reduction by this enzyme with apparent Ki of approximately equal to 0.41 mM.