The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

Cortical astroglia undergo transcriptomic dysregulation in the G93A SOD1 ALS mouse model.

Astroglia are the most abundant glia cell in the central nervous system, playing essential roles in maintaining homeostasis. Key functions of astroglia include, but are not limited to, neurotransmitter recycling, ion buffering, immune modulation, neurotrophin secretion, neuronal synaptogenesis and elimination, and blood-brain barrier maintenance. In neurological diseases, it is well appreciated that astroglia play crucial roles in the disease pathogenesis. In amyotrophic lateral sclerosis (ALS), a motor neuron degenerative disease, astroglia in the spinal cord and cortex downregulate essential transporters, among other proteins, that exacerbate disease progression. Spinal cord astroglia undergo dramatic transcriptome dysregulation. However, in the cortex, it has not been well studied what effects glia, especially astroglia, have on upper motor neurons in the pathology of ALS. To begin to shed light on the involvement and dysregulation that astroglia undergo in ALS, we isolated pure grey-matter cortical astroglia and subjected them to microarray analysis. We uncovered a vast number of genes that show dysregulation at end-stage in the ALS mouse model, G93A SOD1. Many of these genes play essential roles in ion homeostasis and the Wnt-signaling pathway. Several of these dysregulated genes are common in ALS spinal cord astroglia, while many of them are unique. This database serves as an approach for understanding the significance of dysfunctional genes and pathways in cortical astroglia in the context of motor neuron disease, as well as determining regional astroglia heterogeneity, and providing insight into ALS pathogenesis.

3D Printer Generated Tissue iMolds for Cleared Tissue Using Single- and Multi-Photon Microscopy for Deep Tissue Evaluation.

BACKGROUND: Pathological analyses and methodology has recently undergone a dramatic revolution. With the creation of tissue clearing methods such as CLARITY and CUBIC, groups can now achieve complete transparency in tissue samples in nano-porous hydrogels. Cleared tissue is then imagined in a semi-aqueous medium that matches the refractive index of the objective being used. However, one major challenge is the ability to control tissue movement during imaging and to relocate precise locations post sequential clearing and re-staining. METHODS: Using 3D printers, we designed tissue molds that fit precisely around the specimen being imaged. First, images are taken of the specimen, followed by importing and design of a structural mold, then printed with affordable plastics by a 3D printer. RESULTS: With our novel design, we have innovated tissue molds called innovative molds (iMolds) that can be generated in any laboratory and are customized for any organ, tissue, or bone matter being imaged. Furthermore, the inexpensive and reusable tissue molds are made compatible for any microscope such as single and multi-photon confocal with varying stage dimensions. Excitingly, iMolds can also be generated to hold multiple organs in one mold, making reconstruction and imaging much easier. CONCLUSIONS: Taken together, with iMolds it is now possible to image cleared tissue in clearing medium while limiting movement and being able to relocate precise anatomical and cellular locations on sequential imaging events in any basic laboratory. This system provides great potential for screening widespread effects of therapeutics and disease across entire organ systems.

Astroglial transcriptome dysregulation in early disease of an ALS mutant SOD1 mouse model.

Astroglia are a morphologically diverse and highly abundant cell type in the CNS. Despite these obvious observations, astroglia still remain largely uncharacterized at the cellular and molecular level. In disease contexts such as amyotrophic lateral sclerosis (ALS), it has been widely shown that astroglia downregulate crucial physiological functions, become hypertrophied, reactive, and toxic to motor neurons. However, little is known about the astroglia-specific transcriptomic changes that occur during ALS disease progression, especially early in disease. To address this, we FACS-isolated pure astroglia from early and mid-symptomatic superoxide dismutase 1 (SOD1) G93A spinal cord and performed microarray sequencing, in hopes to uncover markers and pathways driving astroglia dysfunction in ALS. After extensive analyses, we uncovered genes selectively enriched and downregulated in both control and SOD1 astroglia at both disease points. In addition, we were able to identify genes and pathways differentially expressed that may have relevance with other neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, suggesting a common theme among astroglial dysfunction in neurodegenerative disease. In aggregate, this study sheds light on the common and unique themes of dysfunction that astroglia undergo during neurodegenerative disease progression and provides candidate targets for therapeutic approaches.

Senolytic and senomorphic secondary metabolites as therapeutic agents in Drosophila melanogaster models of Parkinson's disease.

Drosophila melanogaster is a valuable model organism for a wide range of biological exploration. The well-known advantages of D. melanogaster include its relatively simple biology, the ease with which it is genetically modified, the relatively low financial and time costs associated with their short gestation and life cycles, and the large number of offspring they produce per generation. D. melanogaster has facilitated the discovery of many significant insights into the pathology of Parkinson's disease (PD) and has served as an excellent preclinical model of PD-related therapeutic discovery. In this review, we provide an overview of the major D. melanogaster models of PD, each of which provide unique insights into PD-relevant pathology and therapeutic targets. These models are discussed in the context of their past, current, and future potential use for studying the utility of secondary metabolites as therapeutic agents in PD. Over the last decade, senolytics have garnered an exponential interest in their ability to mitigate a broad spectrum of diseases, including PD. Therefore, an emphasis is placed on the senolytic and senomorphic properties of secondary metabolites. It is expected that D. melanogaster will continue to be critical in the effort to understand and improve treatment of PD, including their involvement in translational studies focused on secondary metabolites.

Novel Genetic Signatures Associated With Sporadic Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a complex polygenetic neurodegenerative disorder. Establishing a diagnosis for ALS is a challenging and lengthy process. By the time a diagnosis is made, the lifespan prognosis is only about two to 5 years. Genetic testing can be critical in assessing a patient's risk for ALS, provided they have one of the known familial genes. However, the vast majority of ALS cases are sporadic and have no known associated genetic signatures. Our analysis of the whole genome sequencing data from ALS patients and healthy controls from the Answer ALS Consortium has uncovered twenty-three novel mutations in twenty-two protein-coding genes associated with sporadic ALS cases. The results show the majority of patients with the sporadic form of ALS have at least one or more mutation(s) in the 22 genes we have identified with probabilities of developing ALS ranging from 25-99%, depending on the number of mutations a patient has among the identified genes. Moreover, we have identified a subset of the ALS cohort that has >17 mutations in the 22 identified. In this case, a patient with this mutation profile has a 99% chance of developing ALS and could be classified as being at high risk for the disease. These genetic biomarkers can be used as an early ALS disease diagnostic tool with a rapid and non-invasive technique.

Neuroglial Senescence, α-Synucleinopathy, and the Therapeutic Potential of Senolytics in Parkinson's Disease.

Parkinson's disease (PD) is the most common movement disorder and the second most prevalent neurodegenerative disease after Alzheimer's disease. Despite decades of research, there is still no cure for PD and the complicated intricacies of the pathology are still being worked out. Much of the research on PD has focused on neurons, since the disease is characterized by neurodegeneration. However, neuroglia has become recognized as key players in the health and disease of the central nervous system. This review provides a current perspective on the interactive roles that α-synuclein and neuroglial senescence have in PD. The self-amplifying and cyclical nature of oxidative stress, neuroinflammation, α-synucleinopathy, neuroglial senescence, neuroglial chronic activation and neurodegeneration will be discussed. Finally, the compelling role that senolytics could play as a therapeutic avenue for PD is explored and encouraged.

Deep Convolutional Neural Networks With Ensemble Learning and Generative Adversarial Networks for Alzheimer's Disease Image Data Classification.

Recent advancements in deep learning (DL) have made possible new methodologies for analyzing massive datasets with intriguing implications in healthcare. Convolutional neural networks (CNN), which have proven to be successful supervised algorithms for classifying imaging data, are of particular interest in the neuroscience community for their utility in the classification of Alzheimer's disease (AD). AD is the leading cause of dementia in the aging population. There remains a critical unmet need for early detection of AD pathogenesis based on non-invasive neuroimaging techniques, such as magnetic resonance imaging (MRI) and positron emission tomography (PET). In this comprehensive review, we explore potential interdisciplinary approaches for early detection and provide insight into recent advances on AD classification using 3D CNN architectures for multi-modal PET/MRI data. We also consider the application of generative adversarial networks (GANs) to overcome pitfalls associated with limited data. Finally, we discuss increasing the robustness of CNNs by combining them with ensemble learning (EL).

Upregulation of Alzheimer's Disease Amyloid-ß Protein Precursor in Astrocytes Both in vitro and in vivo.

BACKGROUND: The amyloid cascade hypothesis of Alzheimer's disease (AD) posits that amyloid-ß (Aß) protein accumulation underlies the pathogenesis of the disease by leading to the formation of amyloid plaques, a pathologic hallmark of AD. Aß is a proteolytic product of amyloid-ß protein precursor (AßPP; APP), which is expressed in both neurons and astrocytes. Although considerable evidence shows that astrocytes may play critical roles in the pathogenesis of AD, the longitudinal changes of amyloid plaques in relationship to AßPP expression in astrocytes and cellular consequences are largely unknown. OBJECTIVE: Here, we aimed to investigate astrocyte-related pathological changes of Aß and AßPP using immunohistochemistry and biochemical studies in both animal and cell models. METHODS/RESULTS: We utilized 5XFAD transgenic mice and found age-dependent upregulation of AßPP in astrocytes demonstrated with astrocytic reactive properties, which followed appearance of amyloid plaques in the brain. We also observed that AßPP proteins presented well-defined punctate immuno reactivity in young animals, whereas AßPP staining showed disrupted structures surrounding amyloid plaques in older mice. Moreover, we utilized astrocyte cell models and showed that pretreatment of Aß42 resulted in downstream astrocyte autonomous changes, including up regulation in AßPP and BACE1 levels, as well as prolonged amyloidogenesis that could be reduced by pharmacological inhibition of BACE1. CONCLUSION: Collectively, our results show that age-dependent AßPP up regulation in astrocytes is a key feature in AD, which will not only provide novel insights for understanding AD progression, but also may offer new therapeutic strategies for treating AD.

Molecularly defined cortical astroglia subpopulation modulates neurons via secretion of Norrin.

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease.

Astrocyte Heterogeneity in the Adult Central Nervous System.

Astrocytes are the most abundant cell type in the central nervous system (CNS), providing critical roles in the overall maintenance and homeostasis. Over 100 years ago, Cajal first showed morphological depictions of different astrocyte populations. Surprisingly, to date astrocytes remain classified in two groups based on their morphological and neuroanatomical positioning. However, accumulating evidence over the past few years is showing that astrocytes are highly diverse throughout the CNS. Astrocyte heterogeneity is not surprisingly, as these cells interact with all other cells in the CNS. Like neurons, astrocytes may also have subpopulations that vary in their functionality. In this mini review, we will explore some of the recent evidence in the adult CNS of astrocyte diversity. First, we will review the very little literature on healthy adult astroglia heterogeneity, followed by the identification of different subpopulations in disease states and how this varies between human and mouse. Exploring this new area of neuroscience will hopefully provide researchers with a new perspective on astrocytes and their heterogeneity throughout the CNS.

Combined adult neurogenesis and BDNF mimic exercise effects on cognition in an Alzheimer's mouse model.

Adult hippocampal neurogenesis (AHN) is impaired before the onset of Alzheimer's disease (AD) pathology. We found that exercise provided cognitive benefit to 5×FAD mice, a mouse model of AD, by inducing AHN and elevating levels of brain-derived neurotrophic factor (BDNF). Neither stimulation of AHN alone, nor exercise, in the absence of increased AHN, ameliorated cognition. We successfully mimicked the beneficial effects of exercise on AD mice by genetically and pharmacologically inducing AHN in combination with elevating BDNF levels. Suppressing AHN later led to worsened cognitive performance and loss of preexisting dentate neurons. Thus, pharmacological mimetics of exercise, enhancing AHN and elevating BDNF levels, may improve cognition in AD. Furthermore, applied at early stages of AD, these mimetics may protect against subsequent neuronal cell death.

Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer's Disease.

Tau is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD), but the mechanism underlying tau-associated neural damage remains unclear. Here, we show that tau can directly interact with nucleoporins of the nuclear pore complex (NPC) and affect their structural and functional integrity. Pathological tau impairs nuclear import and export in tau-overexpressing transgenic mice and in human AD brain tissue. Furthermore, the nucleoporin Nup98 accumulates in the cell bodies of some tangle-bearing neurons and can facilitate tau aggregation in vitro. These data support the hypothesis that tau can directly interact with NPC components, leading to their mislocalization and consequent disruption of NPC function. This raises the possibility that NPC dysfunction contributes to tau-induced neurotoxicity in AD and tauopathies.

Soluble Gamma-secretase Modulators Attenuate Alzheimer's ß-amyloid Pathology and Induce Conformational Changes in Presenilin 1.

A central pathogenic event of Alzheimer's disease (AD) is the accumulation of the Aß42 peptide, which is generated from amyloid-ß precursor protein (APP) via cleavages by ß- and γ-secretase. We have developed a class of soluble 2-aminothiazole γ-secretase modulators (SGSMs) that preferentially decreases Aß42 levels. However, the effects of SGSMs in AD animals and cells expressing familial AD mutations, as well as the mechanism of γ-secretase modulation remain largely unknown. Here, a representative of this SGSM scaffold, SGSM-36, was investigated using animals and cells expressing FAD mutations. SGSM-36 preferentially reduced Aß42 levels without affecting either α- and ß-secretase processing of APP nor Notch processing. Furthermore, an allosteric site was identified within the γ-secretase complex that allowed access of SGSM-36 using cell-based, fluorescence lifetime imaging microscopy analysis. Collectively, these studies provide mechanistic insights regarding SGSMs of this class and reinforce their therapeutic potential in AD.

Astroglia in Thick Tissue with Super Resolution and Cellular Reconstruction.

We utilized the recently published method of passive CLARITY to explore brain astrocytes for the first time with our optimized method. Astrocytes are the fundamental cells in the brain that act to maintain the synaptic activity of neurons, support metabolism of all neurons, and communicate through extensive networks throughout the CNS. They are the defining cell that differentiates lower organisms from humans. From a disease vantage point they are the principal cause of brain tumors and the propagator of neurodegenerative diseases like amyotrophic lateral sclerosis. New methods to study these cells is paramount. Our modified use of CLARITY provides a new way to study these brain cells. To reduce cost, speed up tissue clearing process, reduce human handling error, and to retrieve quantifiable data from single confocal and pseudo-super resolution microscopy we modified and optimized the original protocol.