Common Sleep Disorders in Children.

Childhood sleep disorders can disrupt family dynamics and cause cognitive and behavior problems. Early recognition and management can prevent these complications. Behavior subtypes of childhood insomnias affect 10% to 30% of children and result from inconsistent parental limit-setting and improper sleep-onset association. Behavior insomnias are treated using extinction techniques and parent education. Hypnotic medications are not recommended. Obstructive sleep apnea affects 1% to 5% of children. Polysomnography is required to diagnose obstructive sleep apnea; history and physical examination alone are not adequate. Adenotonsillectomy is the first-line treatment for obstructive sleep apnea. Nasal continuous positive airway pressure is the second-line treatment for children who do not respond to surgery or if adenotonsillectomy is contraindicated. Restless legs syndrome can be difficult to recognize and has an association with attention-deficit/hyperactivity disorder. Management of restless legs syndrome includes treatment of iron deficiency, if identified, and removal of triggering factors. Parasomnias affect up to 50% of children and usually resolve spontaneously by adolescence. Management of parasomnias involves parental education, reassurance, safety precautions, and treating comorbid conditions. Delayed sleep phase syndrome is found during adolescence, manifesting as a night owl preference. Treatment of delayed sleep phase syndrome includes sleep hygiene, nighttime melatonin, and morning bright light exposure. Sleep deprivation is of increasing concern, affecting 68% of people in high school.

Impact of RASSF1A gene methylation on clinico-pathological features of tumor and non-tumor tissue of breast cancer.

BACKGROUND: Breast cancer is the most common malignancy in women caused by genetic and epigenetic changes. Promoter DNA methylation in tumor suppressor gene plays a major role in breast cancer. The study determined the association of promoter DNA methylation of RASSF1A gene with clinicopathological features in tumor and non-tumor tissue. MATERIALS AND METHODS: A cross sectional study was conducted in the Department of Pathology, Government Institute of Medical Sciences, Greater Noida and Molecular Pathology Laboratory, Department of Pathology, Jawaharlal Nehru Medical College, Datta Meghe Institute of Medical Sciences. Two sections, one from tumor and the other from non-tumor tissue, were obtained and processed for DNA extraction and bisulphite conversion. Methylation specific PCR was done and results of RASSF1A promoter methylation were statistically correlated with clinicopathological features. RESULTS: Of the 27 breast cancer tissue, 22 showed invasive ductal carcinoma, one showed invasive lobular carcinoma, another showed ductal carcinoma in situ and three cases showed malignant phyllodes tumor of breast. DNA promoter methylation was found in all the cases. 93% of tumor tissue samples and 67% of the non-tumor tissue samples were found to be aberrantly methylated. Tumor size and histological grade were found to be significantly (p-val <0.05) associated with the RASSF1A gene promoter methylation. CONCLUSION: A significant association of higher tumor size and tumor histological grade with promoter methylation of RASSF1A gene exists suggestive of its being an important determinant of prognostic staging. This critical event in tumorigenesis may be of clinical utility in assessing breast cancer progression. MICRO ABSTRACT: The study focuses on the RASSF1A gene promoter methylation and its impact on the clinicopathological features in Indian breast cancer patients highlighting the differences from other genetically different population. We found that RASFF1A gene methylation has significant impact on tumor size and tumor grade. The work carries high significance because it addresses the DNA methylation of tumor suppressor gene in relevance of breast cancer. It may also be the first such report on Indian patients with breast cancer.

An E3 ubiquitin ligase, cullin-4 regulates retinal differentiation in Drosophila eye.

During organogenesis, cell proliferation is followed by the differentiation of specific cell types to form an organ. Any aberration in differentiation can result in developmental defects, which can result in a partial to a near-complete loss of an organ. We employ the Drosophila eye model to understand the genetic and molecular mechanisms involved in the process of differentiation. In a forward genetic screen, we identified, cullin-4 (cul-4), which encodes an E3 ubiquitin ligase, to play an important role in retinal differentiation. During development, cul-4 is known to be involved in protein degradation, regulation of genomic stability, and regulation of cell cycle. Previously, we have reported that cul-4 regulates cell death during eye development by downregulating Wingless (Wg)/Wnt signaling pathway. We found that loss-of-function of cul-4 results in a reduced eye phenotype, which can be due to onset of cell death. However, we found that loss-of-function of cul-4 also affects retinal development by downregulating retinal determination (RD) gene expression. Early markers of retinal differentiation are dysregulated in cul-4 loss of function conditions, indicating that cul-4 is necessary for differentiation. Furthermore, loss-of-function of cul-4 ectopically induces expression of negative regulators of eye development like Wg and Homothorax (Hth). During eye development, Wg is known to block the progression of a synchronous wave of differentiation referred to as Morphogenetic furrow (MF). In cul-4 loss-of-function background, expression of dpp-lacZ, a MF marker, is significantly downregulated. Our data suggest a new role of cul-4 in retinal differentiation. These studies may have significant bearings on our understanding of early eye development.

Inactivation of Hippo and cJun-N-terminal Kinase (JNK) signaling mitigate FUS mediated neurodegeneration in vivo.

Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. We screened for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly defined deficiencies. We identified hippo (hpo), a component of the evolutionarily conserved Hippo growth regulatory pathway, as a genetic modifier of FUS mediated neurodegeneration. Gain-of-function of hpo triggers cell death whereas its loss-of-function promotes cell proliferation. Downregulation of the Hippo signaling pathway, using mutants of Hippo signaling, exhibit rescue of FUS-mediated neurodegeneration in the Drosophila eye, as evident from reduction in the number of TUNEL positive nuclei as well as rescue of axonal targeting from the retina to the brain. The Hippo pathway activates c-Jun amino-terminal (NH2) Kinase (JNK) mediated cell death. We found that downregulation of JNK signaling is sufficient to rescue FUS-mediated neurodegeneration in the Drosophila eye. Our study elucidates that Hippo signaling and JNK signaling are activated in response to FUS accumulation to induce neurodegeneration. These studies will shed light on the genetic mechanism involved in neurodegeneration observed in ALS and other associated disorders.

miR-277 targets the proapoptotic gene-hid to ameliorate Aß42-mediated neurodegeneration in Alzheimer's model.

Alzheimer's disease (AD), an age-related progressive neurodegenerative disorder, exhibits reduced cognitive function with no cure to date. One of the reasons for AD is the accumulation of Amyloid-beta 42 (Aß42) plaque(s) that trigger aberrant gene expression and signaling, which results in neuronal cell death by an unknown mechanism(s). Misexpression of human Aß42 in the developing retina of Drosophila exhibits AD-like neuropathology. Small non-coding RNAs, microRNAs (miRNAs), post-transcriptionally regulate the expression of their target genes and thereby regulate different signaling pathways. In a forward genetic screen, we identified miR-277 (human ortholog is hsa-miR-3660) as a genetic modifier of Aß42-mediated neurodegeneration. Loss-of-function of miR-277 enhances the Aß42-mediated neurodegeneration. Whereas gain-of-function of miR-277 in the GMR > Aß42 background downregulates cell death to maintain the number of neurons and thereby restores the retinal axonal targeting defects indicating the functional rescue. In addition, gain-of-function of miR-277 rescues the eclosion- and climbing assays defects observed in GMR > Aß42 background. Thus, gain-of-function of miR-277 rescues both structurally as well as functionally the Aß42-mediated neurodegeneration. Furthermore, we identified head involution defective (hid), an evolutionarily conserved proapoptotic gene, as one of the targets of miR-277 and validated these results using luciferase- and qPCR -assays. In the GMR > Aß42 background, the gain-of-function of miR-277 results in the reduction of hid transcript levels to one-third of its levels as compared to GMR > Aß42 background alone. Here, we provide a novel molecular mechanism where miR-277 targets and downregulates proapoptotic gene, hid, transcript levels to rescue Aß42-mediated neurodegeneration by blocking cell death. These studies shed light on molecular mechanism(s) that mediate cell death response following Aß42 accumulation seen in neurodegenerative disorders in humans and provide new therapeutic targets for neurodegeneration.

Signaling interactions among neurons impact cell fitness and death in Alzheimer's disease.

The pathology of Alzheimer's disease involves a long preclinical period, where the characteristic clinical symptoms of the changes in the brain are undetectable. During the preclinical period, homeostatic mechanisms may help prevent widespread cell death. Evidence has pointed towards selective cell death of diseased neurons playing a potentially protective role. As the disease progresses, dysregulation of signaling pathways that govern cell death contributes to neurodegeneration. Aberrant activation of the c-Jun N-terminal kinase pathway has been established in human and animal models of Alzheimer's disease caused by amyloid-beta 42- or tau-mediated neurodegeneration. Clonal mosaic studies in Drosophila that examine amyloid-beta 42 in a subset of neurons suggest complex interplay between amyloid-beta 42-expressing and wild-type cells. This review examines the role of c-Jun N-terminal kinase signaling in the context of cell competition and short-range signaling interactions between amyloid-beta 42-expressing and wild-type neurons. Cell competition is a conserved phenomenon regulating tissue integrity by assessing the fitness of cells relative to their neighbors and eliminating suboptimal cells. Somatic clones of amyloid-beta 42 that juxtapose genetically distinct neuronal cell populations show promise for studying neurodegeneration. Generating genetic mosaics with labeled clones of amyloid-beta 42- or tau-expressing and wild-type neurons will allow us to understand how short-range signaling alterations trigger cell death in neurons and thereby contribute to the progression of Alzheimer's disease. These approaches have the potential to uncover biomarkers for early Alzheimer's disease detection and new therapeutic targets for intervention.

N-Acetyltransferase 9 ameliorates Aß42-mediated neurodegeneration in the Drosophila eye.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, manifests as accumulation of amyloid-beta-42 (Aß42) plaques and intracellular accumulation of neurofibrillary tangles (NFTs) that results in microtubule destabilization. Targeted expression of human Aß42 (GMR > Aß42) in developing Drosophila eye retinal neurons results in Aß42 plaque(s) and mimics AD-like extensive neurodegeneration. However, there remains a gap in our understanding of the underlying mechanism(s) for Aß42-mediated neurodegeneration. To address this gap in information, we conducted a forward genetic screen, and identified N-acetyltransferase 9 (Mnat9) as a genetic modifier of GMR > Aß42 neurodegenerative phenotype. Mnat9 is known to stabilize microtubules by inhibiting c-Jun-N- terminal kinase (JNK) signaling. We found that gain-of-function of Mnat9 rescues GMR > Aß42 mediated neurodegenerative phenotype whereas loss-of-function of Mnat9 exhibits the converse phenotype of enhanced neurodegeneration. Here, we propose a new neuroprotective function of Mnat9 in downregulating the JNK signaling pathway to ameliorate Aß42-mediated neurodegeneration, which is independent of its acetylation activity. Transgenic flies expressing human NAT9 (hNAT9), also suppresses Aß42-mediated neurodegeneration thereby suggesting functional conservation in the interaction of fly Mnat9 or hNAT9 with JNK-mediated neurodegeneration. These studies add to the repertoire of molecular mechanisms that mediate cell death response following accumulation of Aß42 and may provide new avenues for targeting neurodegeneration.

Protocol to study cell death using TUNEL assay in Drosophila imaginal discs.

Cell death maintains tissue homeostasis by eliminating dispensable cells. Misregulation of cell death is seen in diseases like cancer, neurodegeneration, etc. Therefore, cell death assays like TUNEL have become reliable tools, where fragmented DNA of dying cells gets fluorescently labeled and can be detected under microscope. We used TUNEL assay in Drosophila melanogaster third-instar larval eye-antennal imaginal discs to label and quantify cell death. This assay is sensitive to detect DNA fragmentation, an important event, during apoptosis in retinal neurons. For complete details on the use and execution of this profile, please refer to Wang et al. (1999), Tare et al. (2011), and Mehta et al. (2021).

Hippo signaling: bridging the gap between cancer and neurodegenerative disorders.

During development, regulation of organ size requires a balance between cell proliferation, growth and cell death. Dysregulation of these fundamental processes can cause a variety of diseases. Excessive cell proliferation results in cancer whereas excessive cell death results in neurodegenerative disorders. Many signaling pathways known-to-date have a role in growth regulation. Among them, evolutionarily conserved Hippo signaling pathway is unique as it controls both cell proliferation and cell death by a variety of mechanisms during organ sculpture and development. Neurodegeneration, a complex process of progressive death of neuronal population, results in fatal disorders with no available cure to date. During normal development, cell death is required for sculpting of an organ. However, aberrant cell death in neuronal cell population can result in neurodegenerative disorders. Hippo pathway has gathered major attention for its role in growth regulation and cancer, however, other functions like its role in neurodegeneration are also emerging rapidly. This review highlights the role of Hippo signaling in cell death and neurodegenerative diseases and provide the information on the chemical inhibitors employed to block Hippo pathway. Understanding Hippo mediated cell death mechanisms will aid in development of reliable and effective therapeutic strategies in future.

Unbiased automated quantitation of ROS signals in live retinal neurons of Drosophila using Fiji/ImageJ.

Numerous imaging modules are utilized to study changes that occur during cellular processes. Besides qualitative (immunohistochemical) or semiquantitative (Western blot) approaches, direct quantitation method(s) for detecting and analyzing signal intensities for disease(s) biomarkers are lacking. Thus, there is a need to develop method(s) to quantitate specific signals and eliminate noise during live tissue imaging. An increase in reactive oxygen species (ROS) such as superoxide (O2•-) radicals results in oxidative damage of biomolecules, which leads to oxidative stress. This can be detected by dihydroethidium staining in live tissue(s), which does not rely on fixation and helps prevent stress on tissues. However, the signal-to-noise ratio is reduced in live tissue staining. We employ the Drosophila eye model of Alzheimer's disease as a proof of concept to quantitate ROS in live tissue by adapting an unbiased method. The method presented here has a potential application for other live tissue fluorescent images.

Newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye.

Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools poses challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L 2 mutant and GMR-hid, GMR-GAL4 eye. L 2 exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly, newt genes significantly restore missing photoreceptor cells both in L 2 and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.

A Two-Clone Approach to Study Signaling Interactions among Neuronal Cells in a Pre-clinical Alzheimer's Disease Model.

To understand the progression of Alzheimer's disease, studies often rely on ectopic expression of amyloid-beta 42 (Aß42) throughout an entire tissue. Uniform ectopic expression of Aß42 may obscure cell-cell interactions that contribute to the progression of the disease. We developed a two-clone system to study the signaling cross talk between GFP-labeled clones of Aß42-expressing neurons and wild-type neurons simultaneously generated from the same progenitor cell by a single recombination event. Surprisingly, wild-type clones are reduced in size as compared with Aß42-producing clones. We found that wild-type cells are eliminated by the induction of cell death. Furthermore, aberrant activation of c-Jun-N-terminal kinase (JNK) signaling in Aß42-expressing neurons sensitizes neighboring wild-type cells to undergo progressive neurodegeneration. Blocking JNK signaling in Aß42-producing clones restores the size of wild-type clones.

A Positive Feedback Loop of Hippo- and c-Jun-Amino-Terminal Kinase Signaling Pathways Regulates Amyloid-Beta-Mediated Neurodegeneration.

Alzheimer's disease (AD, OMIM: 104300) is an age-related disorder that affects millions of people. One of the underlying causes of AD is generation of hydrophobic amyloid-beta 42 (Aß42) peptides that accumulate to form amyloid plaques. These plaques induce oxidative stress and aberrant signaling, which result in the death of neurons and other pathologies linked to neurodegeneration. We have developed a Drosophila eye model of AD by targeted misexpression of human Aß42 in the differentiating retinal neurons, where an accumulation of Aß42 triggers a characteristic neurodegenerative phenotype. In a forward deficiency screen to look for genetic modifiers, we identified a molecularly defined deficiency, which suppresses Aß42-mediated neurodegeneration. This deficiency uncovers hippo (hpo) gene, a member of evolutionarily conserved Hippo signaling pathway that regulates growth. Activation of Hippo signaling causes cell death, whereas downregulation of Hippo signaling triggers cell proliferation. We found that Hippo signaling is activated in Aß42-mediated neurodegeneration. Downregulation of Hippo signaling rescues the Aß42-mediated neurodegeneration, whereas upregulation of Hippo signaling enhances the Aß42-mediated neurodegeneration phenotypes. It is known that c-Jun-amino-terminal kinase (JNK) signaling pathway is upregulated in AD. We found that activation of JNK signaling enhances the Aß42-mediated neurodegeneration, whereas downregulation of JNK signaling rescues the Aß42-mediated neurodegeneration. We tested the nature of interactions between Hippo signaling and JNK signaling in Aß42-mediated neurodegeneration using genetic epistasis approach. Our data suggest that Hippo signaling and JNK signaling, two independent signaling pathways, act synergistically upon accumulation of Aß42 plaques to trigger cell death. Our studies demonstrate a novel role of Hippo signaling pathway in Aß42-mediated neurodegeneration.

Exploring the efficacy of natural products in alleviating Alzheimer's disease.

Alzheimer's disease (hereafter AD) is a progressive neurodegenerative disorder that affects the central nervous system. There are multiple factors that cause AD, viz., accumulation of extracellular Amyloid-beta 42 plaques, intracellular hyper-phosphorylated Tau tangles, generation of reactive oxygen species due to mitochondrial dysfunction and genetic mutations. The plaques and tau tangles trigger aberrant signaling, which eventually cause cell death of the neurons. As a result, there is shrinkage of brain, cognitive defects, behavioral and psychological problems. To date, there is no direct cure for AD. Thus, scientists have been testing various strategies like screening for the small inhibitor molecule library or natural products that may block or prevent onset of AD. Historically, natural products have been used in many cultures for the treatment of various diseases. The research on natural products have gained importance as the active compounds extracted from them have medicinal values with reduced side effects, and they are bioavailable. The natural products may target the proteins or members of signaling pathways that get altered in specific diseases. Many natural products are being tested in various animal model systems for their role as a potential therapeutic target for AD, and to address questions about how these natural products can rescue AD or other neurodegenerative disorders. Some of these products are in clinical trials and results are promising because of their neuroprotective, anti-inflammatory, antioxidant, anti-amyloidogenic, anticholinesterase activities and easy availability. This review summarizes the use of animal model systems to identify natural products, which may serve as potential therapeutic targets for AD.