Aggregation of E121K mutant D-amino acid oxidase and ubiquitination-mediated autophagy mechanisms leading to amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a terminal adult-onset neuromuscular disorder. Our group has been studying this illness and previously reported novel mutations and rare mutations in a study using next-generation sequencing of DNA samples from Indian ALS patients. In this paper, we focus on the E121K mutation in the DAO gene to understand how it leads to ALS. Our experiments in SH-SY5Y cells indicate that the E121K mutation results in the accumulation of mutant protein aggregates, a change in cell morphology, and the death of neuronal cells. These protein aggregates get ubiquitinated and cause an imbalance in autophagy regulation. We observed an increase in the cellular concentrations of p62, OPTN, and LC3II. Through confocal microscopy studies, we show that the binding of p62 with ubiquitinated aggregates and its recruitment to LC3II mediates autophagosome generation. These relative changes in the key partners in autophagy increase cell death in cells harboring the E121K mutation and is a probable mechanism leading to ALS.

Role of ALS-associated OPTN-K489E mutation in neuronal cell-death regulation.

Optineurin (OPTN) gene is a marker of amyotrophic lateral sclerosis (ALS). However, the role of optineurin protein (OPTN) in ALS pathology is unclear, even though it is known to regulate autophagy, apoptosis, and other survival-death cellular processes. Genetic analysis of Indian ALS patients by our group ascertained a novel mutation K489E in the OPTN gene. To identify the molecular mechanism associated with OPTN and its mutation, we developed an in-vitro cell model using SH-SY5Y cells harbouring OPTN and OPTN-K489E mutation along with its control vector. Since we observed a significant decrease in cell viability in the mutant, we measured the expressions of genes and proteins mediating apoptosis, necroptosis, and autophagy, to establish the role of OPTN in cell death regulation. Our results show that OPTN-K489E mutation changes the relative gene expressions of miRNA-9, REST, CoREST and BDNF, and causes apoptosis. We also observed an up-regulation in the expressions of necroptosis mediated genes RIPK1, RIPK3, and MLKL and autophagy mediated genes TBK1, P62, and LC3II. The results of FACS analyses revealed that this mutation promotes apoptotic and necroptotic processes confirming the pathogenicity of OPTN-K489E.

Chronic social stress blunts core body temperature and molecular rhythms of Rbm3 and Cirbp in mouse lateral habenula.

Chronic social stress in mice causes behavioural and physiological changes that result in perturbed rhythms of body temperature, activity and sleep-wake cycle. To further understand the link between mood disorders and temperature rhythmicity in mice that are resilient or susceptible to stress, we measured core body temperature (Tcore) before and after exposure to chronic social defeat stress (CSDS). We found that Tcore amplitudes of stress-resilient and susceptible mice are dampened during exposure to CSDS. However, following CSDS, resilient mice recovered temperature amplitude faster than susceptible mice. Furthermore, the interdaily stability (IS) of temperature rhythms was fragmented in stress-exposed mice during CSDS, which recovered to control levels following stress. There were minimal changes in locomotor activity after stress exposure which correlates with regular rhythmic expression of Prok2 - an output signal of the suprachiasmatic nucleus. We also determined that expression of thermosensitive genes Rbm3 and Cirbp in the lateral habenula (LHb) were blunted 1 day after CSDS. Rhythmic expression of these genes recovered 10 days later. Overall, we show that CSDS blunts Tcore and thermosensitive gene rhythms. Tcore rhythm recovery is faster in stress-resilient mice, but Rbm3 and Cirbp recovery is uniform across the phenotypes.

A distant angiogenin variant causes amyotrophic lateral sclerosis through loss-of-function mechanisms: Insights from long-timescale atomistic simulations and conformational dynamics.

Amyotrophic Lateral Sclerosis (ALS) is a progressive and incurable neurodegenerative disorder characterized by the degeneration of motor neurons leading to severe muscle atrophy, respiratory failure and death within 3-5 years of disease onset. Missense mutations in Angiogenin (ANG) cause ALS through loss of either ribonucleolytic activity or nuclear translocation activity or both of these functions. Although loss-of-function mechanisms of several rare and ALS-causing ANG variants have been studied before, the structure-function relationship and subsequent functional loss mechanisms of certain novel and uncharacterized rare variants have not been deciphered hitherto. In this study, the structural and dynamic properties of the distantly-located I71V variant, on the functional sites of ANG have been investigated to understand its role in ALS etiology and progression. The I71V variant has a minor allele frequency of <0.06% and thus is classified as a rare variant. Our extensive in silico investigation comprising 1-µs molecular dynamics (MD) simulations, conformational dynamics and related integrated analyses reveal that the I71V variant induces a characteristic conformational switching of catalytic His114 residue resulting in loss of ribonucleolytic activity. Molecular docking and a residue-residue interaction network propagated by an allosteric pathway further support these findings. Moreover, while no conformational alteration of nuclear localization signal governing the nuclear translocation activity was observed, an escalation in mutant plasticity was detected in the structural and essential dynamics simulations. Overall, our study emphasizes that the structure-function relationship of frequently mutating novel ANG variants needs to be established and prioritized in order to advance the pathophysiology and therapeutics of ALS.

Blunted diurnal firing in lateral habenula projections to dorsal raphe nucleus and delayed photoentrainment in stress-susceptible mice.

Daily rhythms are disrupted in patients with mood disorders. The lateral habenula (LHb) and dorsal raphe nucleus (DRN) contribute to circadian timekeeping and regulate mood. Thus, pathophysiology in these nuclei may be responsible for aberrations in daily rhythms during mood disorders. Using the 15-day chronic social defeat stress (CSDS) paradigm and in vitro slice electrophysiology, we measured the effects of stress on diurnal rhythms in firing of LHb cells projecting to the DRN (cellsLHb→DRN) and unlabeled DRN cells. We also performed optogenetic experiments to investigate if increased firing in cellsLHb→DRN during exposure to a weak 7-day social defeat stress (SDS) paradigm induces stress-susceptibility. Last, we investigated whether exposure to CSDS affected the ability of mice to photoentrain to a new light-dark (LD) cycle. The cellsLHb→DRN and unlabeled DRN cells of stress-susceptible mice express greater blunted diurnal firing compared to stress-näive (control) and stress-resilient mice. Daytime optogenetic activation of cellsLHb→DRN during SDS induces stress-susceptibility which shows the direct correlation between increased activity in this circuit and putative mood disorders. Finally, we found that stress-susceptible mice are slower, while stress-resilient mice are faster, at photoentraining to a new LD cycle. Our findings suggest that exposure to strong stressors induces blunted daily rhythms in firing in cellsLHb→DRN, DRN cells and decreases the initial rate of photoentrainment in susceptible-mice. In contrast, resilient-mice may undergo homeostatic adaptations that maintain daily rhythms in firing in cellsLHb→DRN and also show rapid photoentrainment to a new LD cycle.

Identification and characterization of novel and rare susceptible variants in Indian amyotrophic lateral sclerosis patients.

Rare missense variants play a crucial role in amyotrophic lateral sclerosis (ALS) pathophysiology. We report rare/novel missense variants from 154 Indian ALS patients, identified through targeted sequencing of 25 ALS-associated genes. As pathogenic variants could explain only a small percentage of ALS pathophysiology in our cohort, we investigated the frequency of tolerated and benign novel/rare variants, which could be potentially ALS susceptible. These variants were identified in 5.36% (8/149) of sporadic ALS (sALS) cases; with one novel variant each in ERBB4, SETX, DCTN1, and MATR3; four rare variants, one each in PON2 and ANG and two different rare variants in SETX. Identified variants were either absent or present at extremely rare frequencies (MAF < 0.01) in large population databases and were absent in 50 healthy controls sequenced through Sanger method. Furthermore, an oligogenic basis of ALS was observed in three sALS, with co-occurrence of intermediate-length repeat expansions in ATXN2 and a rare/novel variant in DCTN1 and SETX genes. Additionally, molecular dynamics and biochemical functional analysis of an angiogenin variant (R21G) identified from our cohort demonstrated loss of ribonucleolytic and nuclear translocation activities. Our findings suggest that rare variants could be potentially pathogenic and functional studies are warranted to decisively establish the pathogenic mechanisms associated with them.

Rare Angiogenin and Ribonuclease 4 variants associated with amyotrophic lateral sclerosis exhibit loss-of-function: a comprehensive in silico study.

Amyotrophic Lateral Sclerosis (ALS), a debilitating neurodegenerative disorder is related to mutations in a number of genes, and certain genes of the Ribonuclease (RNASE) superfamily trigger ALS more frequently. Even though missense mutations in Angiogenin (ANG) and Ribonuclease 4 (RNASE4) have been previously shown to cause ALS through loss-of-function mechanisms, understanding the role of rare variants with a plausible explanation of their functional loss mechanisms is an important mission. The study aims to understand if any of the rare ANG and RNASE4 variants catalogued in Project MinE consortium caused ALS due to loss of ribonucleolytic or nuclear translocation or both these activities. Several in silico analyses in combination with extensive molecular dynamics (MD) simulations were performed on wild-type ANG and RNASE4, along with six rare variants (T11S-ANG, R122H-ANG, D2E-RNASE4, N26K-RNASE4, T79A-RNASE4 and G119S-RNASE4) to study the structural and dynamic changes in the catalytic triad and nuclear localization signal residues responsible for ribonucleolytic and nuclear translocation activities respectively. Our comprehensive analyses comprising 1.2 µs simulations with a focus on physicochemical, structural and dynamic properties reveal that T11S-ANG, N26K-RNASE4 and T79A-RNASE4 variants would result in loss of ribonucleolytic activity due to conformational switching of catalytic His114 and His116 respectively but none of the variants would lose their nuclear translocation activity. Our study not only highlights the importance of rare variants but also demonstrates that elucidating the structure-function relationship of mutant effectors is crucial to gain insights into ALS pathophysiology and in developing effective therapeutics.

Insights into the role of ribonuclease 4 polymorphisms in amyotrophic lateral sclerosis.

Mutations in certain genes of the Ribonuclease (RNASE) superfamily can cause amyotrophic lateral sclerosis (ALS) through altered RNA processing mechanisms. About 30 of these missense mutations in RNASE5/ANG gene have already been reported in ALS patients. In another gene of the ribonuclease superfamily, ribonuclease 4 (RNASE4), missense mutations and single nucleotide polymorphisms have been identified in patients suffering from ALS. However, their plausible molecular mechanisms of association with ALS are not known. Here, we present the molecular mechanisms of RNASE4 polymorphisms with ALS using all-atom molecular dynamics (MD) simulations followed by functional assay experiments. As most ALS causing mutations in RNASE superfamily proteins affect either the ribonucleolytic or nuclear translocation activity, we examined these functional properties of wild-type and known RNASE4 variants, R10W, A98V, E48D and V75I, using MD simulations. Our simulation predicted that these variants would retain nuclear translocation activity and that E48D would exhibit loss of ribonucleolytic activity, which was subsequently validated by ribonucleolytic assay. Our results give a mechanistic insight into the association of RNASE4 polymorphisms with ALS and show that E48D-RNASE4 would probably be deleterious and cause ALS in individuals harbouring this polymorphism.

Targeted next-generation sequencing reveals novel and rare variants in Indian patients with amyotrophic lateral sclerosis.

Studies on genetic aberrations among Indian amyotrophic lateral sclerosis (ALS) patients are limited to C9orf72 and ATXN2 repeat expansions and mutations in the SOD1 gene. In this study, we used targeted next-generation sequencing to analyze 25 ALS-associated genes in a cohort of 154 Indian ALS patients. We identified known pathogenic mutations in SOD1 (G148D; H44R), TARDBP (M337V; N267S), DAO (R199Q), and ANG (K41I). In addition, we also identified 7 potentially pathogenic missense variants that have not been previously reported in ALS patients; this includes 3 novel variants (OPTN: K489E, DAO: E121K, and SETX: L2163V) that are not reported in large population databases and 4 rare variants (CHMP2B: E45K, SQSTM1: G262R and P438L, ERBB4: R103H) with a minor allele frequency of <0.01 in large population databases. All known pathogenic, novel, and rare variants were detected in only 1 ALS patient each with the exception of the OPTN (K489E) variant that was detected in 2 patients in our cohort. In sum, we identified known and potentially pathogenic novel and rare mutations in 14 (9.1%) ALS patients in our cohort. This study represents the first comprehensive genetic analysis in the ethnically diverse population and thus provides a new insight into the genetics of Indian ALS patients.

C9orf72 hexanucleotide repeat expansions and Ataxin 2 intermediate length repeat expansions in Indian patients with amyotrophic lateral sclerosis.

Repeat expansions in the chromosome 9 open reading frame 72 (C9orf72) gene have been recognized as a major contributor to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in the Caucasian population. Intermediate length repeat expansions of CAG (polyQ) repeat in the ATXN2 gene have also been reported to increase the risk of developing ALS in North America and Europe. We screened 131 ALS patients and 127 healthy controls from India for C9orf72 and ATXN2 repeat expansions. We found pathogenic hexanucleotide expansions in 3 of the 127 sporadic ALS patients, in 1 of the 4 familial ALS patients, and in none of the healthy controls. In addition, our findings suggest that the 10 base-pair deletion that masks detection of C9orf72 repeat expansion does not explain the low frequency of this repeat expansion among Indian ALS patients. Intermediate length polyQ expansions (27Qs-32Qs) in the ATXN2 gene were detected in 6 of the 127 sporadic ALS patients and 2 of the 127 of the healthy controls. Long ATXN2 polyQ repeats (≥33Qs) were not present in any of the ALS patients or controls. Our findings highlight the need for large-scale multicenter studies on Indian ALS patients to better understand the underlying genetic causes.

HSP70 mediates survival in apoptotic cells-Boolean network prediction and experimental validation.

Neuronal stress or injury results in the activation of proteins, which regulate the balance between survival and apoptosis. However, the complex mechanism of cell signaling involving cell death and survival, activated in response to cellular stress is not yet completely understood. To bring more clarity about these mechanisms, a Boolean network was constructed that represented the apoptotic pathway in neuronal cells. FasL and neurotrophic growth factor (NGF) were considered as inputs in the absence and presence of heat shock proteins known to shift the balance toward survival by rescuing pro-apoptotic cells. The probabilities of survival, DNA repair and apoptosis as cellular fates, in the presence of either the growth factor or FasL, revealed a survival bias encoded in the network. Boolean predictions tested by measuring the mRNA level of caspase-3, caspase-8, and BAX in neuronal Neuro2a (N2a) cell line with NGF and FasL as external input, showed positive correlation with the observed experimental results for survival and apoptotic states. It was observed that HSP70 contributed more toward rescuing cells from apoptosis in comparison to HSP27, HSP40, and HSP90. Overexpression of HSP70 in N2a transfected cells showed reversal of cellular fate from FasL-induced apoptosis to survival. Further, the pro-survival role of the proteins BCL2, IAP, cFLIP, and NFκB determined by vertex perturbation analysis was experimentally validated through protein inhibition experiments using EM20-25, Embelin and Wedelolactone, which resulted in 1.27-, 1.26-, and 1.46-fold increase in apoptosis of N2a cells. The existence of a one-to-one correspondence between cellular fates and attractor states shows that Boolean networks may be employed with confidence in qualitative analytical studies of biological networks.