An unnatural amino acid modified human insulin derivative for visual monitoring of insulin aggregation.

Insulin often forms toxic fibrils during production and transportation, which are deposited as amyloids at repeated injection sites in diabetic patients. Distinguishing early fibrils from non-fibrillated insulin is difficult. Herein, we introduce a chemically modified human insulin derivative with a distinct visual colour transition upon aggregation, facilitating insulin quality assessment.

Perinuclear mitochondrial clustering, increased ROS levels, and HIF1 are required for the activation of HSF1 by heat stress.

The heat shock response (HSR) is a conserved cellular defensive response against stresses such as temperature, oxidative stress and heavy metals. A significant group of players in the HSR is the set of molecular chaperones known as heat shock proteins (HSPs), which assist in the refolding of unfolded proteins and prevent the accumulation of damaged proteins. HSP genes are activated by the HSF1 transcription factor, a master regulator of the HSR pathway. A variety of stressors activate HSF1, but the key molecular players and the processes that directly contribute to HSF1 activation remain unclear. In this study, we show that heat shock induces perinuclear clustering of mitochondria in mammalian cells, and this clustering is essential for activation of the HSR. We also show that this perinuclear clustering of mitochondria results in increased levels of reactive oxygen species in the nucleus, leading to the activation of hypoxia-inducible factor-1α (HIF-1α). To conclude, we provide evidence to suggest that HIF-1α is one of the crucial regulators of HSF1 and that HIF-1α is essential for activation of the HSR during heat shock.

Inhibiting Erastin-Induced Ferroptotic Cell Death by Purine-Based Chelators.

Ferroptosis is a cell death event caused by increased lipid peroxidation leading to iron-dependent oxidative stress and is associated with a wide variety of diseases. In recent years, ferroptosis inhibition has emerged as a novel strategy to target different pathologies. Here, we report the synthesis of two purine derivatives, 1 and 2, for iron chelation strategy and evaluate their potency to inhibit erastin-induced ferroptosis. Both compounds showed efficient iron chelation in solution as well as in cellular environment. The crystal structure of the purine derivatives with iron demonstrated a 2 : 1 (ligand to metal center) stoichiometry for iron and purine derivative complexation. The synthesized compounds also decrease the reactive oxygen species concentration in cell cultures. Compound 2 showed better potency towards the prevention of ferroptotic cell death as compared to commercially available iron chelator in the erastin-induced ferroptosis cell culture model. Such purine analogues are potential functional scaffolds for the development of target molecules for ferroptosis inhibition.

Strategies for Interference of Insulin Fibrillogenesis: Challenges and Advances.

The discovery of insulin came with very high hopes for diabetic patients. In 2021, the world celebrated the 100th anniversary of the discovery of this vital hormone. However, external use of insulin is highly affected by its aggregating tendency that occurs during its manufacturing, transportation, and improper handling which ultimately leads to its pharmaceutically and biologically ineffective form. In this review, we aim to discuss the various approaches used for decelerating insulin aggregation which results in the enhancement of its overall structural stability and usage. The approaches that are discussed are broadly classified as either a measure through excipient additions or by intrinsic modifications in the insulin native structure.

Blended polar/nonpolar peptide conjugate interferes with human insulin amyloid-mediated cytotoxicity.

Insulin, a peptide hormone and a key regulator of blood glucose level, is routinely administered to type-I diabetic patients to achieve the required glycemic control. Insulin aggregation and ensuing amyloidosis has been observed at repeated insulin injection sites and in injectable formulations. The latter occurs due to insulin agglomeration during shipping and storage. Such insulin amyloid leads to enhanced immunogenicity and allow potential attachment to cell membranes leading to cell permeability and apoptosis. Small molecule inhibitors provide useful interruption of this process and inhibit protein misfolding as well as amyloid formation. In this context, we report the propensity of a palmitoylated peptide conjugate to inhibit insulin aggregation and amyloid-mediated cytotoxicity, via designed interference with polypeptide interfacial interactions.

Suppression of leptin signaling reduces polyglucosan inclusions and seizure susceptibility in a mouse model for Lafora disease.

Lafora disease (LD) represents a fatal form of neurodegenerative disorder characterized by the presence of abnormally large number of polyglucosan bodies-called the Lafora bodies-in neurons and other tissues of the affected patients. The disease is caused by defects in the EPM2A gene coding for a protein phosphatase (laforin) or the NHLRC1 gene coding for an ubiquitin ligase (malin). Studies have shown that inhibition of glycogen synthesis in the brain could prevent the formation of Lafora bodies in the neurons and reduce seizure susceptibility in laforin-deficient mouse, an established animal model for LD. Since increased glucose uptake is thought to underlie increased glycogen in LD, and since the adipocyte hormone leptin is known to positively regulate the glucose uptake in neurons, we reasoned that blocking leptin signaling might reduce the neuronal glucose uptake and ameliorate the LD pathology. We demonstrate here that mice that were deficient for both laforin and leptin receptor showed a reduction in the glycogen level, Lafora bodies and gliosis in the brain, and displayed reduced susceptibility to induced seizures as compared to animals that were deficient only for laforin. Thus, blocking leptin signaling could be a one of the effective therapeutic strategies in LD.

Human satellite-III non-coding RNAs modulate heat-shock-induced transcriptional repression.

The heat shock response is a conserved defense mechanism that protects cells from physiological stress, including thermal stress. Besides the activation of heat-shock-protein genes, the heat shock response is also known to bring about global suppression of transcription; however, the mechanism by which this occurs is poorly understood. One of the intriguing aspects of the heat shock response in human cells is the transcription of satellite-III (Sat3) long non-coding RNAs and their association with nuclear stress bodies (nSBs) of unknown function. Besides association with the Sat3 transcript, the nSBs are also known to recruit the transcription factors HSF1 and CREBBP, and several RNA-binding proteins, including the splicing factor SRSF1. We demonstrate here that the recruitment of CREBBP and SRSF1 to nSBs is Sat3-dependent, and that loss of Sat3 transcripts relieves the heat-shock-induced transcriptional repression of a few target genes. Conversely, forced expression of Sat3 transcripts results in the formation of nSBs and transcriptional repression even without a heat shock. Our results thus provide a novel insight into the regulatory role for the Sat3 transcripts in heat-shock-dependent transcriptional repression.

Loss of laforin or malin results in increased Drp1 level and concomitant mitochondrial fragmentation in Lafora disease mouse models.

Lafora disease (LD) is an autosomal recessive form of a fatal disorder characterized by the myoclonus epilepsy, ataxia, psychosis, dementia, and dysarthria. A hallmark of LD is the presence of abnormal glycogen inclusions called Lafora bodies in the affected tissues including the neurons. LD can be caused by defects either in the laforin phosphatase coded by the EPM2A gene or in the malin E3 ubiquitin ligase coded by the NHLRC1 gene. The mouse models of LD, created by the targeted disruption of the LD genes, display several neurodegenerative changes. Prominent among them are the autophagic defects, abnormally large lysosomes, neurofibrillary tangles, amyloid beta deposits, and abnormal mitochondria. However, whether or not such neurodegenerative changes are a direct effect of the loss of laforin/malin was not unequivocally established. Here, we show that laforin- or malin-deficient neurons and fibroblasts display a significantly higher number of fragmented mitochondria. Loss of laforin or malin resulted in increased levels of the mitochondrial fission GTPase Drp1, its enhanced mitochondrial targeting, and increased intracellular calcium levels. Intriguingly, laforin and malin display opposite effects on the cellular level of parkin, an ubiquitin ligase of Drp1; loss of laforin led to reduced levels of parkin while the loss of malin resulted in increased parkin levels. Laforin and malin, however, interact with and positively regulate the activity of parkin, thus explaining the molecular basis of increased Drp1 levels in LD tissues. Our results suggest that laforin and malin are novel regulators of mitochondrial quality control pathway and that the mitochondrial dysfunction resulting from the increased Drp1 levels could underlie neuropathology in LD.

Heat shock modulates the subcellular localization, stability, and activity of HIPK2.

The homeodomain-interacting protein kinase-2 (HIPK2) is a highly conserved serine/threonine kinase and is involved in transcriptional regulation. HIPK2 is a highly unstable protein, and is kept at a low level under normal physiological conditions. However, exposure of cells to physiological stress - such as hypoxia, oxidative stress, or UV damage - is known to stabilize HIPK2, leading to the HIPK2-dependent activation of p53 and the cell death pathway. Therefore HIPK2 is also known as a stress kinase and as a stress-activated pro-apoptotic factor. We demonstrate here that exposure of cells to heat shock results in the stabilization of HIPK2 and the stabilization is mediated via K63-linked ubiquitination. Intriguingly, a sub-lethal heat shock (42 °C, 1 h) results in the cytoplasmic localization of HIPK2, while a lethal heat shock (45 °C, 1 h) results in its nuclear localization. Cells exposed to the lethal heat shock showed significantly higher levels of the p53 activity than those exposed to the sub-lethal thermal stress, suggesting that both the level and the nuclear localization are essential for the pro-apoptotic activity of HIPK2 and that the lethal heat shock could retain the HIPK2 in the nucleus to promote the cell death. Taken together our study underscores the importance of HIPK2 in stress mediated cell death, and that the HIPK2 is a generic stress kinase that gets activated by diverse set of physiological stressors.

FoxO3a-mediated autophagy is down-regulated in the laforin deficient mice, an animal model for Lafora progressive myoclonus epilepsy.

Lafora disease (LD) is an autosomal recessive disorder characterized by epileptic seizures, neurodegeneration and accumulation of polyglucosan bodies (Lafora bodies), arising due to defects in either the laforin protein phosphatase or the malin ubiquitin ligase. Among the multiple cellular pathways affected in LD, the specific cause of the autophagy blockade remains unknown. The autophagy impairment however is known to precede the formation of Lafora bodies in the LD mice models. We show here the involvement of a transcription factor, FoxO3a, to be a possible cause for the autophagic defect in cellular and animal models of LD. We find that the expression levels of FoxO3a and its targets Map1LC3b and Atg12 to be at lower levels in laforin-deficient cells and mice. We also find FoxO3a to be regulated indirectly by laforin through the activity of serum/glucocorticoid induced kinase, SGK1. Our results suggest that FoxO3a exerts a negative control over mTOR, and its loss could result in autophagic defects in LD associated with laforin deficiency.

Decreased O-linked GlcNAcylation protects from cytotoxicity mediated by huntingtin exon1 protein fragment.

O-GlcNAcylation is an important post-translational modification of proteins and is known to regulate a number of pathways involved in cellular homeostasis. This involves dynamic and reversible modification of serine/threonine residues of different cellular proteins catalyzed by O-linked N-acetylglucosaminyltransferase and O-linked N-acetylglucosaminidase in an antagonistic manner. We report here that decreasing O-GlcNAcylation enhances the viability of neuronal cells expressing polyglutamine-expanded huntingtin exon 1 protein fragment (mHtt). We further show that O-GlcNAcylation regulates the basal autophagic process and that suppression of O-GlcNAcylation significantly increases autophagic flux by enhancing the fusion of autophagosome with lysosome. This regulation considerably reduces toxic mHtt aggregates in eye imaginal discs and partially restores rhabdomere morphology and vision in a fly model for Huntington disease. This study is significant in unraveling O-GlcNAcylation-dependent regulation of an autophagic process in mediating mHtt toxicity. Therefore, targeting the autophagic process through the suppression of O-GlcNAcylation may prove to be an important therapeutic approach in Huntington disease.

Lafora disease proteins laforin and malin negatively regulate the HIPK2-p53 cell death pathway.

Lafora disease (LD) is an autosomal recessive, progressive, and fatal form of a neurodegenerative disorder characterized by the presence of Lafora polyglucosan bodies. LD is caused by defects in either the laforin protein phosphatase or the malin E3 ubiquitin ligase. Laforin and malin were shown play key roles in proteolytic processes, unfolded stress response, and glycogen metabolism. Therefore, the LD proteins laforin and malin are thought to function as pro-survival factors and their loss thus could result in neurodegeneration. To understand the molecular pathway leading to the cell death in LD, in the present study, we investigated the possible role of LD proteins in the p53-mediated cell death pathway. We show that loss of laforin or malin results in the increased level and activity of p53, both in cellular and animal models of LD, and that this is primarily due to the increased levels of Hipk2, a proapoptotic activator of p53. Overexpression of laforin or malin confers protection against Hipk2-mediated cell death by targeting the Hipk2 to the cytoplasmic compartment. Taken together, our study strengthens the notion that laforin and malin are pro-survival factors, and that the activation of Hipk2-p53 cell death pathway might underlie neurodegeneration in LD.

Dysfunctions in endosomal-lysosomal and autophagy pathways underlie neuropathology in a mouse model for Lafora disease.

Lafora progressive myoclonus epilepsy (also known as Lafora disease, LD) is an inherited and fatal form of a neurodegenerative disorder characterized by the presence of carbohydrate-rich inclusions called Lafora bodies. LD can be caused by defects in the laforin phosphatase or the malin ubiquitin ligase and the clinical symptoms resulting from these two defects are almost similar. In order to understand the molecular basis of LD pathogenesis and the role of Lafora bodies in neuropathology, we have studied the laforin-deficient mice as a model and show here that Lafora bodies recruit proteasomal subunit, endoplasmic reticulum chaperone GRP78/Bip, autophagic protein p62 and endosomal regulators Rab5 and Rab7. The laforin-deficient brain also reveals the proliferation of enlarged lysosomes, lipofuscin granules, amyloid-ß peptides and increased levels of insoluble form of ubiquitinated protein, indicating a significant impairment in the cellular degradative pathway. Further, abnormal dendrites and increased gliosis, especially at the vicinity of Lafora bodies, were noted in the LD brain. Taken together, our study suggests that the neuropathology in LD is not limited to Lafora bodies, that some of the neuropathological changes in LD are likely to be secondary effects caused by Lafora bodies, and that impairment in the autophagy-endosomal-lysosomal pathways might underlie some of the symptoms in LD.

Lafora progressive myoclonus epilepsy: Disease mechanism and therapeutic attempts.

Lafora disease (LD) is a life-threatening autosomal recessive and progressive neurodegenerative disorder that primarily affects adolescents, resulting in mortality within a decade of onset. The symptoms of LD include epileptic seizures, ataxia, dementia, and psychosis. The underlying pathology involves the presence of abnormal glycogen inclusions in neurons and other tissues, which may contribute to neurodegeneration. LD is caused by loss-of-function mutations in either the EPM2A gene or the NHLRC1 gene. These two genes, respectively, code for laforin phosphatase and malin ubiquitin ligase, and are thought to function, as a functional complex, in diverse cellular pathways. One of the major pathways affected in LD is glycogen metabolism; defects here lead to abnormally higher levels of glycogen and its hyperphosphorylation and aggregation, resulting in the formation of Lafora inclusion bodies. Currently, there is no effective therapy for LD. Studies, particularly from animal models, provide distinct insights into the fundamental mechanisms of diseases and potential avenues for therapeutic interventions. The purpose of this review is to present a comprehensive overview of our current knowledge regarding the disease, its genetics, the animal models that have been developed, and the therapeutic strategies that are being developed based on an understanding of the disease mechanism.

Thermocapillary migration of a drop with a thermally conducting stagnant cap.

Hypothesis The thermocapillary migration of a spherical drop with a stagnant cap in the presence of a constant applied temperature gradient can be strongly affected by the finite thermal conductivity of the stagnant cap. Numerics The heat conduction of the stagnant cap is analytically modeled. The effects of the additional interfacial stresses generated by the disturbances to the local temperature field due to the presence of the cap at the fluid-fluid interface and the corresponding velocity of migration of the drop are evaluated by solving for the temperature and hydrodynamic field equations in and around the drop. An asymptotic model is derived to predict the terminal velocity in the presence of an infinitely conducting stagnant cap. Findings The effects of the surface conductivity and size of the stagnation region alongside the bulk thermal conductivities and viscosities of the drop and surrounding media are evaluated. The terminal velocity of the drop is shown to have a monotonic dependence on the conductivity of the stagnant cap. The bounds to the terminal velocity increment due to the stagnant cap are derived. These bounds can be of significance to multiphysics problems involving particle laden drops, Pickering emulsions and other multi-phase technologies where the conductivity of the surface adsorbents is non-negligible.

Retinal vascular pathology in a mouse model of Lafora progressive myoclonus epilepsy.

Neurodegenerative diseases (ND) affect distinct populations of neurons and manifest various clinical and pathological symptoms. A subset of ND prognoses has been linked to vascular risk factors. Consequently, the current study investigated retinal vascular abnormalities in a murine model of Lafora neurodegenerative disease (LD), a fatal and genetic form of progressive myoclonus epilepsy that affects children. Here, arterial rigidity was evaluated by measuring pulse wave velocity and vasculature deformations in the retina. Our findings in the LD mouse model indicate altered pulse wave velocity, retinal vascular thinning, and convoluted retinal arteries.

Malin and laforin are essential components of a protein complex that protects cells from thermal stress.

The heat-shock response is a conserved cellular process characterized by the induction of a unique group of proteins known as heat-shock proteins. One of the primary triggers for this response, at least in mammals, is heat-shock factor 1 (HSF1)--a transcription factor that activates the transcription of heat-shock genes and confers protection against stress-induced cell death. In the present study, we investigated the role of the phosphatase laforin and the ubiquitin ligase malin in the HSF1-mediated heat-shock response. Laforin and malin are defective in Lafora disease (LD), a neurodegenerative disorder associated with epileptic seizures. Using cellular models, we demonstrate that these two proteins, as a functional complex with the co-chaperone CHIP, translocate to the nucleus upon heat shock and that all the three members of this complex are required for full protection against heat-shock-induced cell death. We show further that laforin and malin interact with HSF1 and contribute to its activation during stress by an unknown mechanism. HSF1 is also required for the heat-induced nuclear translocation of laforin and malin. This study demonstrates that laforin and malin are key regulators of HSF1 and that defects in the HSF1-mediated stress response pathway might underlie some of the pathological symptoms in LD.

The SCN1A gene variants and epileptic encephalopathies.

The voltage-gated sodium channels are fundamental units that evoke the action potential in excitable cells such as neurons. These channels are integral membrane proteins typically consisting of one α-subunit, which forms the larger central pore of the channel, and two smaller auxiliary ß-subunits, which modulate the channel functions. Genetic alterations in the SCN1A gene coding for the α-subunit of the neuronal voltage-gated sodium ion channel, type 1 (NaV 1.1), is associated with a spectrum of seizure-related disorders in human, ranging from a relatively milder form of febrile seizures to a more severe epileptic condition known as the Dravet syndrome. Among the epilepsy genes, the SCN1A gene perhaps known to have the largest number of disease-associated alleles. Here we present a meta-analysis on the SCN1A gene variants and provide comprehensive information on epilepsy-associated gene variants, their frequency, the predicted effect on the protein, the ethnicity of the affected along with the inheritance pattern and the associated epileptic phenotype. We also summarize our current understanding on the pathophysiology of the SCN1A gene defects, disease mechanism, genetic modifiers and their clinical and diagnostic relevance.

Identification and characterization of novel splice variants of the human EPM2A gene mutated in Lafora progressive myoclonus epilepsy.

The EPM2A gene, defective in the fatal neurodegenerative disorder Lafora disease (LD), is known to encode two distinct proteins by differential splicing; a phosphatase active cytoplasmic isoform and a phosphatase inactive nuclear isoform. We report here the identification of three novel EPM2A splice variants with potential to code for five distinct proteins in alternate reading frames. These novel isoforms, when ectopically expressed in cell lines, show distinct subcellular localization, interact with and serve as substrates of malin ubiquitin ligase-the second protein defective in LD. Two phosphatase active isoforms interact to form a heterodimeric complex that is inactive as a phosphatase in vitro, suggesting an antagonistic function for laforin isoforms if expressed endogenously in significant amounts in human tissues. Thus alternative splicing could possibly be one of the mechanisms by which EPM2A may regulate the cellular functions of the proteins it codes for.