Amyloid beta is critically involved in the initiation and progression of Alzheimer’s disease (AD). This peptide is neurotoxic. It can cause cell death by acting on neurons. It can also cause neuronal cell death by acting on glial cells, resulting in the production of toxic and inflammatory molecules, which eventually cause neurotoxicity. In this study, we show that safranal, a compound present in the spice saffron, reduces reactive oxygen species (ROS) and nitric oxide (NO) levels from hippocampal neuronal cells, HT22, treated with oligomeric amyloid beta. It reduces alteration in mitochondrial membrane potential induced by amyloid beta. Safranal shows protective effects against amyloid beta-induced toxicity not only to HT22 cells but also to human neurons. Furthermore, we show that safranal reduces ROS, NO and inflammatory molecule levels from amyloid beta-treated astrocytic cells. Importantly, it provides protection to human neurons from the toxic effects of conditioned medium derived from human astrocytes treated with amyloid beta. Thus, safranal protects neurons against amyloid beta-induced direct and indirect toxicity, suggesting that it could be beneficial in the fight against AD.

Iron accumulation and mitochondrial dysfunction in astroglia are reported in Parkinson’s disease (PD). Astroglia control iron availability in neurons in which dopamine (DA) synthesis is affected in PD. Despite their intimate relationship the role of DA in astroglial iron homeostasis is limited. Here we show that DA degrades iron storage protein ferritin in astroglial cells involving lysosomal proteolysis. Lysosomal ferritinophagy is mainly associated with macroautophagy; however, we revealed the involvement of chaperone-mediated autophagy (CMA) in DA-induced ferritin degradation. In CMA, cytosolic proteins containing a specific pentapeptide motif bind with HSC70 to be transported to lysosome mediated by LAMP2A. We identified the conserved pentapeptide motif in ferritin-H (Ft-H), mutations of which resulted loss of its interaction with HSC70. Pharmacological inhibitors of HSC70 or LAMP2/2A knockdown blocks DA-induced Ft-H degradation. DA also induces cytosolic cargo NCOA4 for ferritinophagy. We further reveal that DA promotes cathepsin B to lysis ferritin within the lysosome. Inhibitor of cathepsin B, knocking down of LAMP2, or HSC70 inhibitor attenuate DA-induced elevated mitochondrial iron level. Our results establish a direct role of DA on astroglial iron homeostasis and novel involvement of CMA in ferritin degradation in response to a biological stimulus. These results also may help in better understanding iron dyshomeostasis and mitochondrial dysfunction reported in PD.

The blood-brain barrier (BBB) is a specialized semipermeable structure that restricts the exchange of materials between central nervous system parenchyma and blood vessels. Nanocarriers have shown the potential to cross the BBB via receptor-mediated transcytosis (RMT) or adsorption-mediated transcytosis (AMT), though efficiency remains limited. Here, we report two different polymer nanomicelles of 20-60 nm size that efficiently cross the BBB via either RMT or AMT. We have tested their performance in a transwell-based model, tumor spheroid model, and in vivo mice model. We found that lower numbers of receptors per nanomicelle are critical for RMT and direct membrane penetration property of nanomicelle is critical for AMT. Transcytosis can be enhanced by 8-10 times using the optimized nanomicelles that leads to 8 times enhanced penetration in tumor spheroid and 26-35 times enhanced delivery to deep brain tissue. These polymer nanomicelles can be used as nanocarrier for in vivo delivery of therapeutic biomolecules in brain.

The molecular basis of neuronal damage in SARS-CoV-2 infection remains elusive. Here, we identify the viral accessory protein Orf6 as a key driver of necroptotic neuronal death. Through a systematic screen of 22 structural and non-structural SARS-CoV-2 proteins, Orf6 emerged to be the most cytotoxic to human cells. In primary human neurons, Orf6 selectively activates the necroptotic pathway by inducing phosphorylation of RIPK3 and MLKL, without engaging apoptosis, pyroptosis, or autophagy. These findings were corroborated in SARS-CoV-2-infected neurons and in hippocampal brain sections from COVID-19 patients, where elevated necroptosis markers were observed. Mechanistically, Orf6 was found to directly interact with the mitochondrial membrane protein Mitochondrial Carrier Homolog 1 (MTCH1), triggering mitochondrial calcium overload, membrane potential loss, and oxidative stress. Genetic knockdown of MTCH1 or pharmacological inhibition of RIPK3 effectively rescued neurons from Orf6-induced necroptosis and mitochondrial dysfunction. Collectively, our findings reveal a novel, mitochondria-dependent mechanism of viral neuropathogenesis and implicate Orf6-mediated necroptosis as a contributor to long-term neurological sequelae in COVID-19.

Neuroblastoma is the most common extra-cranial solid tumor diagnosed mostly in children below the age of five years and comprises of about 15% of all paediatric cancer deaths. Tumor initiating cancer stem cells (CSCs) can be targeted for better treatment approaches. BASP1-AS1 is a long non coding (Lnc) RNA that is a divergent LncRNA for its coding gene brain abundant membrane attached signal protein 1 (BASP1). We had earlier demonstrated it to be expressed in foetus derived human neural progenitor cells (hNPCs), where it was a positive regulator of BASP1 and was critical for neural differentiation. In this study, we have investigated the role of BASP1-AS1 in CSCs derived from the human neuroblastoma cell line SH-SY5Y. We cultured SH-SY5Y cells on Poly-D-Lysine coated flasks in serum free media supplemented with growth factors, which led to the enrichment of CSCs as determined by marker expression. When grown on ultra-low attachment flasks, these cells formed CSCs enriched neurospheres. We examined the effects of BASP1-AS1 siRNA mediated knockdown on CSCs enriched SH-SY5Y cells and SH-SY5Y derived neurospheres. BASP1-AS1 knockdown decreased the levels of the corresponding gene BASP1 and the rate of cell proliferation of CSCs enriched cells along with low expression of Ki67. It also reduced the mRNA levels of stem cell and pluripotency gene markers (CD133, CD44, c-KIT, SOX2, OCT4 and NANOG), as also Wnt 2 and the Wnt pathway effector β catenin. It also abrogated the formation of neurospheres in ultra-low attachment flasks. A similar effect on proliferation and stemness related properties was seen on BASP1 knockdown. BASP1-AS1 and its related pathways may provide a point of intervention for the CSCs population in neuroblastoma.

Lactate Dehydrogenase A (LDH-A) catalyzes the conversion of pyruvate into lactate in the last step of glycolysis, and its heightened expression is associated with the enhanced glycolytic metabolism in solid and hematopoietic cancers. LDH-A plays an essential role in the survival and pluripotency of hematopoietic stem cells (HSCs) via sustained glycolysis. Loss of LDH-A reduced HSCs mitochondrial fitness, maintenance, and repopulation ability. Activated T cells rely on glycolysis to engage in effector functions however the role for LDH-A in CD8+ T cell differentiation is unclear. We generated mice with conditional targeting of the LDH-A allele (LDH-Af/f) and crossed them with CD4-Cre to selectively delete LDH-A in T cells (LDH-Af/fCD4-Cre). To generate LDH-A deficient antigen-specific CD8+ T cells, we backcrossed LDH-Af/fCD4-Cre mice with OTI-transgenic mice bearing a TCR recognizing Ovalbumin peptide (Ova257-264) presented by H-2Kb, and generated OTI/ LDH-Af/fCD4-Cre (OTI/LDH-A-/-) mice. Antigen-specific T cells from OTI or OTI/LDH-A-/- mice were adoptively transferred into syngeneic recipients followed by immunization with Ova-expressing Listeria monocytogenes (Lm-OVA). During the effector phase, 8 days after infection, OTI/LDH-A-/- T cells had diminished expansion compared to OTI cells and displayed a T memory (TMEM)-like signature with expansion of memory precursors, higher expression of CD127 and lower expression of KLRG1. Gene expression analysis showed that LDH-A deficient T cells were enriched for genes encoding factors known to identify TMEM CD8+ cells, including Bcl6, Tcf7 and its targets Eomes and Id3, and the Klf2 targets CCR7 and CD62L. In contrast, transcripts encoding regulators of T effector (TEFF) differentiation such as Prdm1, T-bet, KLRG1, IFNγ were enriched in control OTI cells. The expression of key enzymes involved in lipolysis and FAO were also significantly different. LDH-A deficient cells displayed increased expression of Cpt1a, the rate-limiting enzyme involved in transport of fatty acids into the mitochondria for β-oxidation. These cells also had elevated transcripts encoding regulators of lipolysis, Lal and Atgl, and the glycerol transporter aquaporin 9 (Aqp9), involved in TMEM development. Metabolomics analysis showed high levels of metabolic intermediates of glycolysis and TCA cycle. During the memory phase, 60 days after infection with Lm-Ova, there were no numerical differences in TMEM subsets in recipients of OTI and OTI/LDH-A-/- T cells. We sorted TMEM cells and adoptively transferred them into new hosts, which were subsequently rechallenged with Lm-OVA. LDH-A-/- deficient TMEM cells were functionally superior compared to their OTI counterparts as determined by reduced CFUs per spleen in hosts receiving OTI/LDH-A-/- T cells. Rechallenged OTI/LDH-A-/- TMEM cells also produced higher amounts of IFNg, and contained a greater proportion of stem-like memory T cells (TSCM). Given these findings, we sought to determine if this superior TMEM phenotype would confer greater anti-tumor immunity. We injected syngeneic hosts with B16-F10 melanoma expressing OVA (B16-OVA), then transferred OTI or OTI/LDH-A-/- T cells and monitored tumor growth. Strikingly, we found significantly larger tumors, a higher CD4/8 T cell ratio, and less CD8+ T cell infiltration in recipients of OTI/LDH-A-/- T cells. OTI/LDH-A-/- T cells within the tumor had higher expression of the immunosuppressive protein ICOS, and hallmark features of T cell exhaustion including higher expression of TIM-3, and lower levels of Slamf6 (Ly108) and CD62L. These results indicate that selective ablation of LDH-A in CD8+ T cells imprints a TMEM-like phenotype but impairs TEFF differentiation and function within the tumor microenvironment compromising anti-tumor immunity. Our findings uncover a new role of LDH-A in CD8+ TEFF vs. TMEM responses and indicate that LDH-A has a decisive role in regulating T cell differentiation and functionality.

Oligodendrocytes (OL) are the myelinating cells of the central nervous system that mediate nerve conduction. Loss of oligodendrocytes results in demyelination, triggering neurological deficits. Developing a better understanding of the cell signaling pathways influencing OL development may aid in the development of therapeutic strategies. The primary focus of this study was to investigate and elucidate the cell signaling pathways implicated in the developmental maturation of oligodendrocytes using human fetal neural stem cells (hFNSCs)–derived primary OL and MO3.13 cell line. Successful differentiation into OL was established by examining morphological changes, increased expression of mature OL markers MBP, MOG and decreased expression of pre-OL markers CSPG4 and O4. Analyzing transcriptional datasets (using RNA sequencing) in pre-OL and mature OL derived from hFNSCs revealed the novel and critical involvement of the JAK-STAT cell signaling pathway in terminal OL maturation. The finding was validated in MO3.13 cell line whose differentiation was accompanied by upregulation of IL-6 and the transcription factor STAT3. Increased phosphorylated STAT3 (pY705) levels were demonstrated by western blotting in hFNSCs-derived primary OL as well as terminal maturation in MO3.13 cells, thus validating the involvement of the JAK-STAT pathway in OL maturation. Pharmacological suppression of STAT3 phosphorylation (confirmed by western blotting) was able to prevent the increase of MBP-positive cells as demonstrated by flow cytometry. These novel findings highlight the involvement of the JAK-STAT pathway in OL maturation and raise the possibility of using this as a therapeutic strategy in demyelinating diseases.

Clearing of toxic huntingtin protein aggregates from neuron cells is critical for treatment of Huntington's disease. However, this is very challenging and requires the control of intracellular processes and associated biochemical activities. Here, we report the designed nanoparticle-based clearing of toxic huntingtin protein aggregates via cellular autophagy induction. We have designed five different nanoparticles of 15-40 nm size that are composed of an iron oxide/zinc oxide/carbon core and a surface terminated with hemin/amine/arginine/trehalose. These nanoparticles induce cellular autophagy by three different mechanisms, and all of them clear toxic huntingtin aggregates from neuron cells. Such clearance of huntingtin aggregates by nanoparticles has shown a substantial increase in cell viability typically up to 7-fold. Our result suggests that autophagy-inducing nanodrugs may be designed for clearing toxic amyloid aggregates from the brain and treatment of neurodegenerative diseases.

Neuroblastomas are pediatric tumors arising from undifferentiated cells of neural crest origin with stem cell-like characteristics. Dysregulation of Wnt/β-catenin signaling has been shown to be linked to the development of various tumors. Activated Wnt signaling results in β-catenin accumulation in the nucleus to support pro-neoplastic traits. DKK1, a secreted glycoprotein, is an inhibitor of Wnt signaling, and the addition of DKKI to the culture medium has been used to suppress the Wnt pathway. This study aimed to analyze the role of Dickopff-1 as a potential differentiating agent for the neuroblastoma cell line SH-SY5Y and neurospheres derived from it. The treatment of SH-5Y5Y derived neurospheres by DKK1 resulted in their disintegration and reduced proliferation markers like Ki67, PCNA. DKK1 treatment to the neurospheres also resulted in the loss of cancer stem cell markers like CD133, KIT and pluripotency markers like SOX2, OCT4, NANOG. DKK1 treatment caused reduction in mRNA expression of β-catenin and TCF genes like TCF4, TCF12. When the SHSY5Y cancer cells were grown under differentiating conditions, DKKI caused neuronal differentiation by itself, and in synergy with retinoic acid. This was verified by the expression of markers like MAPT, DCX, GAP43, ENO2 and also with changes in neurite length. We concluded that Wnt inhibition, as exemplified by DKK1 treatment, is therefore a possible differentiating condition and also suppresses the proliferative and cancer stemness related properties of SH-SY5Y neuroblastoma cells.

STAT1 (Signal Transducer and Activator of Transcription 1), belongs to the STAT protein family, essential for cytokine signaling. Ithas been reported to have either context dependent oncogenic or tumor suppressor roles in different tumors. Earlier, we demonstrated that Glioblastoma multiforme (GBMs) overexpressing FAT1, an atypical cadherin, had poorer outcomes. Overexpressed FAT1 promotes pro-tumorigenic inflammation, migration/invasion by downregulating tumor suppressor gene, PDCD4. Here, we demonstrate that STAT1 is a novel mediator downstream to FAT1, in downregulating PDCD4 in GBMs. In-silico analysis of GBM databases as well as q-PCR analysis in resected GBM tumors showed positive correlation between STAT1 and FAT1 mRNA levels. Kaplan-Meier analysis showed poorer survival of GBM patients having high FAT1 and STAT1 expression. SiRNA-mediated knockdown of FAT1 decreased STAT1 and increased PDCD4 expression in glioblastoma cells (LN229 and U87MG). Knockdown of STAT1 alone resulted in increased PDCD4 expression. In silico analysis of the PDCD4 promoter revealed four putative STAT1 binding sites (Site1-Site4). ChIP assay confirmed the binding of STAT1 to site1. ChIP-PCR revealed decrease in the binding of STAT1 on the PDCD4 promoter after FAT1 knockdown. Site directed mutagenesis of Site1 resulted in increased PDCD4 luciferase activity, substantiating STAT1 mediated PDCD4 inhibition. EMSA confirmed STAT1 binding to the Site 1 sequence. STAT1 knockdown led to decreased expression of pro-inflammatory cytokines and EMT markers, and reduced migration/invasion of GBM cells. This study therefore identifies STAT1 as a novel downstream mediator of FAT1, promoting pro-tumorigenic activity in GBM, by suppressing PDCD4 expression.

Hypoxic insult to the fetal brain causes loss of vulnerable premyelinating oligodendrocytes and arrested oligodendrocyte differentiation. Astrocytes influence oligodendrocyte differentiation and the astrocytic response to hypoxia could affect oligodendrocyte maturation under hypoxia. To identify pathways by which astrocytes influence oligodendroglial maturation in hypoxic injury, human fetal neural stem cell-derived astrocytes were exposed to 0.2 % oxygen for 48 hours. Transcriptomic analysis revealed the upregulation of the cholesterolbiosynthesis pathway in hypoxia-exposed astrocytes. Hypoxia-exposed primary astrocytes and astrocytic cell line (SVG) showed increased expression of hydroxy-methyl-glutaryl-CoA reductase (HMGCR), squalene epoxidase (SQLE), apolipoprotein E (apoE) and ATP-binding cassette transporter 1 (ABCA1) on qPCR and Western blot. Hypoxic SVG also showed increased cholesterol content in cells and culture supernatants and increased cell surface expression of ABCA1. Interestingly hypoxia-exposed premyelinating oligodendrocytes (Mo3.13) showed reduced cholesterol along with decreased expression of HMGCR and SQLE on qPCR and Western blot. Exogenous cholesterol increased the differentiation of Mo3.13 as measured by increased expression of myelin basic protein (MBP) on flow cytometry. Hypoxia exposure resulted in increased cholesterol transport from astrocytes to oligodendrocytes in cocultures with BODIPY-cholesterol labelled SVG and membrane-labelled Mo3.13. As exogenous cholesterol enhanced oligodendrocyte differentiation, our findings indicate that increased cholesterol synthesis by astrocytes and transport to oligodendrocytes could supplement oligodendroglial maturation in conditions of hypoxic brain injury in neonates.

NO-mediated regulation of PDL-1 in cancer cells.

Cerebral ischemic stroke and glioma are the two leading causes of patient mortality globally. Despite physiological variations, 1 in 10 people who have an ischemic stroke go on to develop brain cancer, most notably gliomas. In addition, glioma treatments have also been shown to increase the risk of ischemic strokes. Stroke occurs more frequently in cancer patients than in the general population, according to traditional literature. Unbelievably, these events share multiple pathways, but the precise mechanism underlying their co-occurrence remains unknown. Transcription factors (TFs), the main components of gene expression programmes, finally determine the fate of cells and homeostasis. Both ischemic stroke and glioma exhibit aberrant expression of a large number of TFs, which are strongly linked to the pathophysiology and progression of both diseases. The precise genomic binding locations of TFs and how TF binding ultimately relates to transcriptional regulation remain elusive despite a strong interest in understanding how TFs regulate gene expression in both stroke and glioma. As a result, the importance of continuing efforts to understand TF-mediated gene regulation is highlighted in this review, along with some of the primary shared events in stroke and glioma.

Although subcellular targeting can enhance the therapeutic performance of most drugs, such targeting requires appropriate carrier-based delivery that can bypass endosomal/lysosomal trafficking. Recent works show that nanocarriers can be designed for direct cell membrane translocation and nonendocytic uptake, bypassing the usual endocytosis processes. Here we show that this approach can be adapted for the rapid cell nucleus delivery of molecular drugs. In particular, a guanidinium-terminated nanocarrier is used to create a weak interaction-based carrier-drug nanoassembly for direct membrane translocation into the cytosol. The rapid and extensive entry of a drug-loaded nanocarrier into the cell without any vesicular coating and affinity of the drug to the nucleus allows their nucleus labeling. Compared to endocytotic uptake that requires more than hours for cell uptake followed by predominant lysosomal entrapment, this nonendocytic uptake labels the nucleus within a few minutes without any lysosomal trafficking. This approach may be utilized for nanocarrier-based subcellular targeting of drugs for more effective therapy.

LDH-A deletion resets tumor microenvironment.

The Zika virus (ZIKV) outbreaks and its co-relation with microcephaly have become a global health concern. It is primarily transmitted by a mosquito, but can also be transmitted from an infected mother to her fetus causing impairment in brain development, leading to microcephaly. However, the underlying molecular mechanism of ZIKV-induced microcephaly is poorly understood. In this study, we explored the role of ZIKV non-structural protein NS4A and NS4B in ZIKV pathogenesis in a well-characterized primary culture of human fetal neural stem cells (fNSCs). We observed that the co-transfection of NS4A and NS4B altered the neural stem cell fate by arresting proliferation and inducing premature neurogenesis. NS4A + NS4B transfection in fNSCs increased autophagy and dysregulated notch signaling. Further, it also altered the regulation of downstream genes controlling cell proliferation. Additionally, we reported that 3 methyl-adenine (3-MA), a potent autophagy inhibitor, attenuated the deleterious effects of NS4A and NS4B as evidenced by the rescue in Notch1 expression, enhanced proliferation, and reduced premature neurogenesis. Our attempts to understand the mechanism of autophagy induction indicate the involvement of mitochondrial fission and ROS. Collectively, our findings highlight the novel role of NS4A and NS4B in mediating NSC fate alteration through autophagy-mediated notch degradation. The study also helps to advance our understanding of ZIKV-induced neuropathogenesis and suggests autophagy as a potential target for anti-ZIKV therapeutic intervention.

Zika virus (ZIKV) infection during the first trimester of the pregnancy may lead to Congenital zika syndrome in the neonates. The viral infection hampers foetal brain development and causes microcephaly. Human neural progenitor cells (hNPCs) play an important role in brain development, however they are highly susceptible to ZIKV infection. In this study, we elucidated the molecular mechanisms that lead to cellular alterations in hNPCs due to ZIKV E-protein. We investigated proliferation, differentiation, migration and inflammation in hNPCs, which may lead to microcephaly. In our study, we found that ZIKV E-protein causes cell cycle arrest, decrease in proliferation and increase in mitotic length of the dividing hNPCs. We observed CyclinD1 and upstream molecules (p21 and p53) of the pathway are dysregulated, and intracellular calcium at basal level as well as upon ATP stimulation were reduced following over expression of ZIKV E-protein. ZIKV E-protein transfected hNPCs exhibited pre-mature differentiation with pro-neural genes upregulated. Furthermore, ZIKV E-protein disrupted migrational properties of hNPCs and caused elevated levels of inflammatory chemokines and cytokines. To gain insights into molecular mechanisms of these effects on hNPCs, we explored the possible involvement of long non coding RNAs in ZIKV neuropathogenesis. We have shortlisted lncRNAs associated with differentially expressed genes from publicly available transcriptomic data and found some of those lncRNAs are differentially expressed upon E-protein transfection of hNPCs. Gene ontology analysis suggest these lncRNAs play an important role in regulation of viral life cycle, host's defence response and cell proliferation.

PDF file - 2219K, CO enhances DNA damage of PCa cells.