Vibha Taneja

Toxic species in amyloid disorders: Oligomers or mature fibrils

Protein aggregation is the hallmark of several neurodegenerative disorders. These protein aggregation (fibrillization) disorders are also known as amyloid disorders. The mechanism of protein aggregation involves conformation switch of the native protein, oligomer formation leading to protofibrils and finally mature fibrils. Mature fibrils have long been considered as the cause of disease pathogenesis; however, recent evidences suggest oligomeric intermediates formed during fibrillization to be toxic. In this review, we have tried to address the ongoing debate for these toxic amyloid species. We did an extensive literature search and collated information from Pubmed (http://www.ncbi.nlm.nih.gov) and Google search using various permutations and combinations of the following keywords: Neurodegeneration, amyloid disorders, protein aggregation, fibrils, oligomers, toxicity, Alzheimer′s Disease, Parkinson′s Disease. We describe different instances showing the toxicity of mature fibrils as well as oligomers in Alzheimer′s Disease and Parkinson′s Disease. Distinct structural framework and morphology of amyloid oligomers suggests difference in toxic effect between oligomers and fibrils. We highlight the difference in structure and proposed toxicity pathways for fibrils and oligomers. We also highlight the evidences indicating that intermediary oligomeric species can act as potential diagnostic biomarker. Since the formation of these toxic species follow a common structural switch among various amyloid disorders, the protein aggregation events can be targeted for developing broad-range therapeutics. The therapeutic trials based on the understanding of different protein conformers (monomers, oligomers, protofibrils and fibrils) in amyloid cascade are also described.

Curcumin Prevents Formation of Polyglutamine Aggregates by Inhibiting Vps36, a Component of the ESCRT-II Complex

Small molecules with antioxidative properties have been implicated in amyloid disorders. Curcumin is the active ingredient present in turmeric and known for several biological and medicinal effects. Adequate evidence substantiates the importance of curcumin in Alzheimers disease and recent evidence suggests its role in Prion and Parkinsons disease. However, contradictory effects have been suggested for Huntingtons disease. This difference provided a compelling reason to investigate the effect of curcumin on glutamine-rich (Q-rich) and non-glutamine-rich (non Q-rich) amyloid aggregates in the well established yeast model system. Curcumin significantly inhibited the formation of htt72Q-GFP (a Q-rich) and Het-s-GFP (a non Q-rich) aggregates in yeast. We show that curcumin prevents htt72Q-GFP aggregation by down regulating Vps36, a component of the ESCRT-II (Endosomal sorting complex required for transport). Moreover, curcumin disrupted the htt72Q-GFP aggregates that were pre-formed in yeast and cured the yeast prion, [PSI +].

Integration Host Factor of Mycobacterium tuberculosis, mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.

Identification of L84F mutation with a novel nucleotide change c.255G > T in the superoxide dismutase gene in a North Indian family with amyotrophic lateral sclerosis

Abstract Mutations in the superoxide dismutase (SOD1) gene account for ∼15% and in the transactive response DNA binding protein (TARDBP) gene for ∼5% of familial amyotrophic lateral sclerosis (FALS) cases. These two genes were analysed in two siblings from North India with ALS and a positive family history. The coding region of SOD1 and TARDBP genes was sequenced in both siblings. Genetic variation identified in SOD1 was typed in unaffected family members (n = 11), sporadic ALS patients (n = 48) and healthy controls (n = 35). Molecular dynamic (MD) simulations were performed on wild-type (WT) and mutant monomers of SOD1 to determine structural changes due to the identified mutation. A novel heterozygous nucleotide variation (c.255G > T) was identified in exon 4 of SOD1 in the two siblings and two asymptomatic family members but not in SALS patients and healthy controls. This variation results in a known non-synonymous substitution from leucine to phenylalanine at position 84 (L84F), making it a triallelic variation. Large conformational changes were observed in the zinc loop and electrostatic loop in an L84F mutant compared to WT SOD1 in MD simulations. In conclusion, this is the first report of mutation in SOD1 associated with FALS in India. Structural perturbations in L84F SOD1 may cause dimer destabilization, with decreased metal affinity leading to oligomerization.

Analysis of C9orf72 repeat expansion in amyotrophic lateral sclerosis patients from North India

Pathogenic expansion of a hexanucleotide repeat in C9orf72 is associated with ~30% of familial ALS and ~7% of sporadic ALS patients amongst different populations. This repeat expansion was screened in 75 ALS patients and 115 healthy individuals from North India. On analysis by repeat-primed PCR, pathogenic expansion was not observed either in ALS patients or healthy controls. These observations are similar to the findings in most of the Asian populations.

Expression analysis of protein homeostasis pathways in the peripheral blood mononuclear cells of sporadic amyotrophic lateral sclerosis patients

Misfolded protein aggregates are the hallmark of Amyotrophic Lateral Sclerosis (ALS) which suggests involvement of protein homeostasis pathways in etiology of ALS. However, status of protein homeostasis in peripheral blood of ALS is not well established. We analyzed expression levels of key genes of proteostasis pathways in peripheral blood mononuclear cells (PBMCs) of sporadic ALS (sALS) patients and healthy controls. Increased protein carbonylation was observed in patients reflecting oxidative damage in PBMCs. We observed increased transcript and protein levels of GRP78 suggesting Endoplasmic reticulum (ER) insult to cells. Further, significant upregulation of spliced XBP1 and two stress sensors: IRE1α/ERN1 and ATF6 indicated induction of unfolded protein response (UPR). Genes involved in autophagosome initiation (ULK1, ULK2, ATG13); nucleation and elongation (BECLIN1, ATG7, ATG16L1, ATG5, ATG10) and vesicular trafficking genes were significantly increased in patients. Increased lipidation of LC3 validated induction of autophagy. Accumulation of low molecular weight ubiquitinated proteins in patients suggested deregulation of proteasome (UPS) pathway. In addition, cytosolic chaperones (HSP70 and HSP27) and HSF1 were elevated in patients. Increased TDP43 indicated role of TDP43 in disease pathology. Our findings suggest that there is oxidative insult and upregulation of UPR, vesicular trafficking and autophagy in PBMCs of sALS patients.

Q-Rich Yeast Prion [PSI+] Accelerates Aggregation of Transthyretin, a Non-Q-Rich Human Protein

Interactions amongst different amyloid proteins have been proposed as a probable mechanism of aggregation and thus an important risk factor for the onset as well as progression of various neurodegenerative disorders including Alzheimers, Parkinsons, Huntingtons, and Amyotrophic Lateral Sclerosis. Evidences suggest that transthyretin (TTR), a plasma protein associated with transthyretin amyloidosis or familial polyneuropathy (FAP) interacts with heterologous amyloid proteins including amyloid beta and islet amyloid polypeptide. In addition, recent clinical studies have revealed the presence of systemic polyneuropathy associated with FAP mutations in patients with spinocerebral ataxia, amyotrophic lateral sclerosis, and new familial systematic prion disease. Hence, it is important to investigate the interactions amongst different amyloid proteins to gain better insight into the pathology of amyloid disorders. Yeast has been an excellent model system to study interaction/ cross-seeding between heterologous amyloid proteins, more because of presence of endogenous yeast prions. Here, we examined interactions of non-glutamine (non-Q)-rich transthyretin, with glutamine (Q)-rich yeast prion protein Sup35. We established aggregation of an engineered double (F87M/L110M) mutant M-TTR-GFP in yeast. This mutant is monomeric and readily formed aggregates compared to WT-TTR-GFP in yeast at acidic pH. Interestingly, aggregation of M-TTR-GFP was significantly enhanced in presence of [PSI+], an endogenous prion form of Sup35. Different variants of [PSI+] seeded M-TTR-GFP with different efficiencies and curing of [PSI+] (losing the prion form) in these strains reduced aggregation. Moreover, overexpression of prion domain of Sup35 fused to RFP (NM-RFP) also increased M-TTR-GFP aggregation. M-TTR-GFP and NM-RFP aggregates co-localized in perivacuolar and juxtranuclear region. Sup35 protein was even immunocaptured in M-TTR-GFP aggregates. However, M-TTR-GFP overexpression did not induce Sup35 aggregation. Thus, it appears to be a unidirectional interaction between these two amyloid proteins. However, no affect on M-TTR-GFP aggregation was observed due to another yeast prion, [PIN+]. Our findings thus show the molecular interaction of transthyretin with yeast prion and support that sequence similarity is not the prime requirement for heterologous amyloid interactions.

Identification of prognostic and susceptibility markers in chronic myeloid leukemia using next generation sequencing

Background Incidence of Chronic Myeloid Leukemia (CML) is continuously increasing and expected to reach 100,000 patients every year by 2030. Though the discovery of Imatinib Mesylate (IM) has brought a paradigm shift in CML treatment, 20% patients show resistance to this tyrosine kinase inhibiter (TKI). Therefore, it is important to identify markers, which can predict the occurrence and prognosis of CML. Clinical Exome Sequencing, panel of more than 4800 genes, was performed in CML patients to identify prognostic and susceptibility markers in CML. Methods Enrolled CML patients (n=18) were segregated as IM responders (n=10) and IM failures (n=8) as per European Leukemia Net (ELN), 2013 guidelines. Healthy controls (n=5) were also enrolled. DNA from blood of subjects was subjected to Next Generation Sequencing. Rare mutations present in one patient group and absent in another group were considered as prognostic markers, whereas mutations present in more than 50% patients were considered as susceptibility markers. Result Mutations in genes associated with cancer related functions were found in different patient groups. Four variants: rs116201358, rs4014596, rs52897880 and rs2274329 in C8A, UNC93B1, APOH and CA6 genes, respectively, were present in IM responders; whereas rs4945 in MFGE8 was present in IM failures. Mutations in HLA-DRB1 (rs17878951), HLA-DRB5 (rs137863146), RPHN2 (rs193179333), CYP2F1 (rs116958555), KCNJ12 (rs76684759) and FUT3 (rs151218854) were present as susceptibility markers. Conclusion The potential genetic markers discovered in this study can help in predicting response to IM as frontline therapy. Susceptibility markers may also be used as panel for individuals prone to have CML.