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Histopathological analysis (A–J) and molecular evaluation of hSOD1G93A signal (B) in the sciatic nerve and Schwann cell samples of 60-day-old presymptomatic SOD1G93A and aged paired wild-type mice. Immunofluorescence staining of MAP2 (A,B; red), GAP-43 (C,D; green), S100 (E,F; green) and p75NGF (G,H; green) in the sciatic nerve of 60-day-old presymptomatic SOD1G93A mice (B,D,F,H) and their wild-type controls (A,C,E,G). MAP2 and GAP-43 are markers of neuronal fibers; S100 and p75NGF are markers of Schwann cells. Cell nuclei were stained with DAPI (blue). The insert boxes in the bottom left of images show a higher magnification of the cell profiles. Methylene blue staining of Schwann cell myelin sheets of sciatic nerve of 60-day-old presymptomatic SOD1G93A mice (J) and their wild-type controls (I) are also seen. Scale bars: 10 μm. Of note, the same staining pattern was observed for both genotypes (SOD1G93A and wild-type controls) for all cell markers and for the histological sections. Representative bands of PCR for specific gene markers of human SOD1G93A (hSOD1G93A) and actin b (Actb) in sciatic nerve (K) and Schwann cells enriched samples (L) obtained by flow cytometry sorting of SOD1G93A and wild-type control mice.
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Schwann cells are the main source of paracrine support to motor neurons. Oxidative stress and mitochondrial dysfunction have been correlated to motor neuron death in Amyotrophic Lateral Sclerosis (ALS). Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown...
Citations
... Within the cluster of CNV-driven genes in SALS2, we found a large number of new candidate ALS genes mainly implicated in oxidative and inflammatory signaling cascades [52][53][54][55][56][57] . Among these, identification of gain and overexpression of TRAF2 was in accordance with previous evidence that correlated elevated expression levels of this gene with inflammatory processes in PD and other neurological disorders 58,59 . On the contrary, copy number loss and reduced expression levels HSPA5 in SALS2 may reflect the suppression of neuroprotective role of this molecule against ER stress-associated cell death, leading to oxidative stress and alterations in calcium homeostasis, and rendering neurons vulnerable to degeneration [60][61][62][63][64] . ...
Amyotrophic lateral sclerosis (ALS) is an incurable and fatal neurodegenerative disease. Increasing the chances of success for future clinical strategies requires more in-depth knowledge of the molecular basis underlying disease heterogeneity. We recently laid the foundation for a molecular taxonomy of ALS by whole-genome expression profiling of motor cortex from sporadic ALS (SALS) patients. Here, we analyzed copy number variants (CNVs) occurring in the same patients, by using a customized exon-centered comparative genomic hybridization array (aCGH) covering a large panel of ALS-related genes. A large number of novel and known disease-associated CNVs were detected in SALS samples, including several subgroup-specific loci, suggestive of a great divergence of two subgroups at the molecular level. Integrative analysis of copy number profiles with their associated transcriptomic data revealed subtype-specific genomic perturbations and candidate driver genes positively correlated with transcriptional signatures, suggesting a strong interaction between genomic and transcriptomic events in ALS pathogenesis. The functional analysis confirmed our previous pathway-based characterization of SALS subtypes and identified 24 potential candidates for genomic-based patient stratification. To our knowledge, this is the first comprehensive “omics” analysis of molecular events characterizing SALS pathology, providing a road map to facilitate genome-guided personalized diagnosis and treatments for this devastating disease.
... Sporadic disease represents 90% of ALS patients and iPSCs from these patients have been differentiated into neurons that exhibited cytoplasmic aggregation of TDP-43 [44]. Furthermore, gene expression profiling of sporadic ALS iPSC-derived MNs suggests deficiencies in mitochondrial function [45]. Additionally, familial ALS mutation phenotypes have been recapitulated in iPSC disease models showing characteristic proteinopathies, endoplasmic reticulum stress and oxidative stress [42,46]. ...
Neurological diseases such as Alzheimer's disease and Parkinson's disease are growing problems, as average life expectancy is increasing globally. Drug discovery for neurological disease remains a major challenge. Poor understanding of disease pathophysiology and incomplete representation of human disease in animal models hinder therapeutic drug development. Recent advances with induced pluripotent stem cells (iPSCs) have enabled modeling of human diseases with patient-derived neural cells. Utilizing iPSC-derived neurons advances compound screening and evaluation of drug efficacy. These cells have the genetic backgrounds of patients that more precisely model disease-specific pathophysiology and phenotypes. Neural cells derived from iPSCs can be produced in a large quantity. Therefore, application of iPSC-derived human neurons is a new direction for neuronal drug discovery.
... Importantly, considering sporadic disease represents 90% of ALS patients, sporadic iPSCs have also been differentiated into neurons and were shown to exhibit TDP-43 pathology (Burkhardt et al., 2013;Qian et al., 2017). Furthermore, gene expression profiling of sporadic ALS iPSCMNs suggested deficits in mitochondrial function (Alves et al., 2015). More studies are required to better understand the pathogenesis of sporadic disease, and human cell culture models are currently the only way to gain insight into what triggers and propagates sporadic ALS and FTD. ...
Neurological diseases, including dementias such as Alzheimer's disease (AD) and fronto-temporal dementia (FTD) and degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS), are responsible for an increasing fraction of worldwide fatalities. Researching these heterogeneous diseases requires models that endogenously express the full array of genetic and epigenetic factors which may influence disease development in both familial and sporadic patients. Here, we discuss the two primary methods of developing patient-derived neurons and glia to model neurodegenerative disease: reprogramming somatic cells into induced pluripotent stem cells (iPSCs), which are differentiated into neurons or glial cells, or directly converting (DC) somatic cells into neurons (iNeurons) or glial cells. Distinct differentiation techniques for both models result in a variety of neuronal and glial cell types, which have been successful in displaying unique hallmarks of a variety of neurological diseases. Yield, length of differentiation, ease of genetic manipulation, expression of cell-specific markers, and recapitulation of disease pathogenesis are presented as determining factors in how these methods may be used separately or together to ascertain mechanisms of disease and identify therapeutics for distinct patient populations or for specific individuals in personalized medicine projects.
Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron death due to nuclear loss and cytoplasmic aggregation of the splice factor TDP-43. Pathologic TDP-43 associates with stress granules (SGs) and downregulating the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASO) prolongs survival in the TAR4/4 sporadic ALS mouse model, a strategy now in clinical trials. Here, we used AAV-mediated RNAi delivery to achieve lasting and targeted Atxn2 knockdown after a single injection. To achieve this, a novel AAV with improved transduction potency of our target cells was used to deliver Atxn2 -targeting miRNAs. Mouse dosing studies demonstrated 55% Atxn2 knockdown in frontal cortex and 25% knockdown throughout brainstem and spinal cord after intracerebroventricular injection at a dose 40x lower than used in other recent studies. In TAR4/4 mice, miAtxn2 treatment increased mean and median survival by 54% and 45% respectively (p<0.0003). Mice showed robust improvement across strength-related measures ranging from 24-75%. Interestingly, treated mice showed increased vertical activity above wildtype, suggesting unmasking of an FTD phenotype with improved strength. Histologically, lower motor neuron survival improved with a concomitant reduction in CNS inflammatory markers. Additionally, phosphorylated TDP-43 was reduced to wildtype levels. Bulk RNA sequencing revealed correction of 153 genes in the markedly dysregulated transcriptome of mutant mice, several of which are described in the human ALS literature. In slow progressing hemizygous mice, treatment rescued weight loss and improved gait at late time points. Cumulatively the data support the utility of AAV-mediated RNAi against Atxn2 as a robust and translatable treatment strategy for sporadic ALS.
Background and objectives:
Induced pluripotent stem cells (iPSCs) derived three-dimensional (3D) model for rare neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) is emerging as a novel alternative to human diseased tissue to explore the disease etiology and potential drug discovery. In the interest of the same, we have generated a TDP-43-mutated human iPSCs (hiPSCs) derived 3D organoid model of ALS disease. The high-resolution mass spectrometry (MS)-based proteomic approach is used to explore the differential mechanism under disease conditions and the suitability of a 3D model to study the disease.
Materials and methods:
The hiPSCs cell line was procured from a commercial source, grown, and characterized following standard protocols. The mutation in hiPSCs was accomplished using CRISPR/Cas-9 technology and predesigned gRNA. The two groups of organoids were produced by normal and mutated hiPSCs and subjected to the whole proteomic profiling by high-resolution MS in two biological replicates with three technical replicas of each.
Results:
The proteomic analysis of normal and mutated organoids revealed the proteins associated with pathways of neurodegenerative disorders, proteasomes, autophagy, and hypoxia-inducible factor-1 signaling. Differential proteomic analysis revealed that the mutation in TDP-43 gene caused proteomic deregulation, which impaired protein quality mechanisms. Furthermore, this impairment may contribute to the generation of stress conditions that may ultimately lead to the development of ALS pathology.
Conclusion:
The developed 3D model represents the majority of candidate proteins and associated biological mechanisms altered in ALS disease. The study also offers novel protein targets that may uncloud the precise disease pathological mechanism and be considered for future diagnostic and therapeutic purposes for various neurodegenerative disorders.
The spinal cord (SC) links brain and body, mediates movement, autonomic function and relays sensory information. SC anatomical organization is modular. The central grey matter is organized into: the dorsal horn that contains sensory neurons that transmit information from the periphery to the brain and the ventral horn that contains Motorneurons (MNs) that transmit motor commands from the CNS to muscle. The surrounding white matter contains nerve fibers of the ascending and descending tracts. MNs die in motor neuron diseases (MNDs). The development, specialization and function of spinal cord circuits rely heavily on the expression of homeoprotein (HPs).HPs bind DNA and regulate gene transcription and contain a DNA binding motif. The homeodomain (HD) is highly conserved suggesting evolutionary preservation of function. In the nervous system HPs are involved in brain regionalization, cell differentiation, synaptogenesis and neuronal survival. Many HPs transfer to neighboring cells to regulate transcription and protein translation non-cell autonomously. The first question addressed is whether neuroprotection is a general HP characteristic. I tested whether different HPs protect against neuronal cell death and DNA damage caused by H2O2. ENGRAILED1 (EN1) increased survival and reduced DNA damage. HPs of different classes and species promoted survival of embryonic brain cells. EN internalization and high affinity DNA binding are required for neuroprotection. We then extended this to human LUHMES and the oxidative stressor, 6-OHDA. These results suggest that neuroprotection is a common property of HPs effective against different oxidative stressors.Inhibitory interneurons that synapse directly on motorneurons (MNs) are the only cells that express EN1 in the SC. The number of SC neurons is the same in EN1-heterozygous mice and WT littermates at 1 and 3 months old (mo). At 4.5mo, EN1-het mice have about 20% fewer αMNs. The significant reduction in αMNs is associated with muscle weakness and spinal reflex deficits. The degeneration of αMNs progresses, and at 15.5mo less than 50% remain in the EN1-het mouse. The innervation of the neuromuscular junction (NMJ) of 3mo EN1-het mice is significantly reduced and this precedes the loss of large αMNs. Thus, mice with reduced EN1 have early and progressive muscle weakness and abnormal spinal reflexes that are accompanied by NMJ denervation and later loss of large αMNs.Exogenous recombinant human EN1 prevented the EN1-het phenotype. Intrathecal injection of hEN1 was made at 3mo when behavioral deficits and NMJ abnormalities are observed but there is no αMN loss. A single injection of hEN1 restores muscle strength, normalizes spinal reflex, prevents NMJ denervation and promotes the survival of αMNs. Additional experiments show the restorative and neuroprotective effect of hEN1 lasts several months, but has no beneficial effect if administered after more than 40% of αMNs are lost and that high-affinity DNA binding is required for neuroprotection. Experiments designed to neutralize extracellular EN1 in the spinal cord suggest that there is a non-cell autonomous component for EN1 activity in MN physiology.As an initial proof of concept for therapy, human iPSCs were differentiated into spinal MNs. After 5 days in vitro differentiated MNs start to die, but when hEN1 is added, MNs survived for at least 15 days. Moreover, hEN1 was able to protect MNs derived from Amyotrophic Lateral Sclerosis (ALS) patients carrying the SOD1 A4V and C9ORF72 mutations against oxidative stress in vitro.This work shows that all HPs tested in vitro promote the survival of embryonic neurons and hEN1 protects human LUHMES and MNs. In vivo reduction of EN1 results in the progressive loss of αMNs, accompanied by motor signs and NMJ abnormalities. All are restored when exogenous hEN1 is provided. These results suggest that hEN1 may have therapeutic value in MNDs such as ALS.
Myelin sheath is critical for the proper functioning of the peripheral nervous system (PNS), which allows the effective conduction of nerve impulses. Fibroblast growth factor 9 (FGF9) is an autocrine and paracrine protein in the fibroblast growth factor family that regulates cell differentiation and proliferation. Fgf9 Schwann cell (SC) conditional knockout mice were developed to detect the role of FGF9 in the PNS. In our study, the absence of Fgf9 led to delayed myelination in early development. The expression of mature SC‐related genes decreased, and the expression of genes associated with immature SCs increased in the Fgf9 knockout mice. These data were consistent with the morphology and praxeology we observed during the development of the peripheral nerves. Extracellular‐regulated kinases 1/2 (ERK1/2) are key signals for myelination, and our results showed that Fgf9 ablation led to the inactivation of ERK1/2. Further research was performed to detect the role of FGF9 in peripheral nerve injury. In superoxide dismutase 1‐G93A mice with Fgf9 SC knockout, we found that Fgf9 ablation inhibited the expressions of Cd68, Il‐1β, and Cd86, which contributed to the degeneration of the axon and myelin sheath. In our study, the absence of Fgf9 led to delayed myelination in early development. Further research was performed to detect the role of FGF9 in peripheral nerve injury. We found that Fgf9 ablation inhibited the expressions of Cd68, Il‐1β, and Cd86, which contributed to the degeneration of the axon and myelin sheath.
Emerging evidence indicates there is a complex interplay between metabolism and chronic disorders in the nervous system. In particular, the pyruvate dehydrogenase (PDH) kinase (PDK)–lactic acid axis is a critical link that connects metabolic reprogramming and the pathophysiology of neurological disorders. PDKs, via regulation of PDH complex activity, orchestrate the conversion of pyruvate either aerobically to acetyl-CoA, or anaerobically to lactate. The kinases are also involved in neurometabolic dysregulation under pathological conditions. Lactate, an energy substrate for neurons, is also a recently acknowledged signaling molecule involved in neuronal plasticity, neuron–glia interactions, neuroimmune communication, and nociception. More recently, the PDK–lactic acid axis has been recognized to modulate neuronal and glial phenotypes and activities, contributing to the pathophysiologies of diverse neurological disorders. This review covers the recent advances that implicate the PDK–lactic acid axis as a novel linker of metabolism and diverse neuropathophysiologies. We finally explore the possibilities of employing the PDK–lactic acid axis and its downstream mediators as putative future therapeutic strategies aimed at prevention or treatment of neurological disorders.
