Human Amyotrophic Lateral Sclerosis Research Articles

Bridging the Gap: Harnessing Plant Bioactive Molecules to Target Gut Microbiome Dysfunctions in Amyotrophic Lateral Sclerosis.

The correlation between neurodegenerative diseases and the gut microbiome is increasingly evident, with amyotrophic lateral sclerosis (ALS) being particularly notable for its severity and lack of therapeutic options. The gut microbiota, implicated in the pathogenesis and development of ALS, plays a crucial role in the disease. Bioactive plant molecules, specifically volatile compounds in essential oils, offer a promising therapeutic avenue due to their anti-inflammatory properties and gut-modulating effects. Our narrative review aimed to identify microbiota-associated bacteria in ALS and analyze the benefits of administering bioactive plant molecules as much-needed therapeutic options in the management of this disease. A comprehensive search of PubMed database articles published before December 2023, encompassing research on cell, human, and animal ALS models, was conducted. After selecting, analyzing, and discussing key articles, bacteria linked to ALS pathogenesis and physiopathology were identified. Notably, positively highlighted bacteria included Akkermansia muciniphila (Verrucomicrobia phylum), Faecalibacterium prausnitzii, and Butyrivibrio spp. (Firmicutes phylum). Conversely, members of the Escherichia coli spp. (Proteobacteria phylum) and Ruminococcus spp. (Firmicutes phylum) stood out negatively in respect to ALS development. These bacteria were associated with molecular changes linked to ALS pathogenesis and evolution. Bioactive plant molecules can be directly associated with improvements in the microbiome, due to their role in reducing inflammation and oxidative stress, emerging as one of the most promising natural agents for enriching present-day ALS treatments.

Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferon-gamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.

PAD2 dysregulation and aberrant protein citrullination feature prominently in reactive astrogliosis and myelin protein aggregation in sporadic ALS

Alteration in protein citrullination (PC), a common posttranslational modification (PTM), contributes to pathogenesis in various inflammatory disorders. We previously reported that PC and protein arginine deiminase 2 (PAD2), the predominant enzyme isoform that catalyzes this PTM in the central nervous system (CNS), are altered in mouse models of amyotrophic lateral sclerosis (ALS). We now demonstrate that PAD2 expression and PC are altered in human postmortem ALS spinal cord and motor cortex compared to controls, increasing in astrocytes while trending lower in neurons. Furthermore, PC is enriched in protein aggregates that contain the myelin proteins PLP and MBP in ALS. These results confirm our findings in ALS mouse models and suggest that altered PAD2 and PC contribute to neurodegeneration in ALS.

Reduction of inflammation and mitochondrial degeneration in mutant SOD1 mice through inhibition of voltage-gated potassium channel Kv1.3.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no effective therapy, causing progressive loss of motor neurons in the spinal cord, brainstem, and motor cortex. Regardless of its genetic or sporadic origin, there is currently no cure for ALS or therapy that can reverse or control its progression. In the present study, taking advantage of a human superoxide dismutase-1 mutant (hSOD1-G93A) mouse that recapitulates key pathological features of human ALS, we investigated the possible role of voltage-gated potassium channel Kv1.3 in disease progression. We found that chronic administration of the brain-penetrant Kv1.3 inhibitor, PAP-1 (40 mg/Kg), in early symptomatic mice (i) improves motor deficits and prolongs survival of diseased mice (ii) reduces astrocyte reactivity, microglial Kv1.3 expression, and serum pro-inflammatory soluble factors (iii) improves structural mitochondrial deficits in motor neuron mitochondria (iv) restores mitochondrial respiratory dysfunction. Taken together, these findings underscore the potential significance of Kv1.3 activity as a contributing factor to the metabolic disturbances observed in ALS. Consequently, targeting Kv1.3 presents a promising avenue for modulating disease progression, shedding new light on potential therapeutic strategies for ALS.

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

EphrinB2 knockdown in cervical spinal cord preserves diaphragm innervation in a mutant SOD1 mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron loss. Importantly, non-neuronal cell types such as astrocytes also play significant roles in disease pathogenesis. However, mechanisms of astrocyte contribution to ALS remain incompletely understood. Astrocyte involvement suggests that transcellular signaling may play a role in disease. We examined contribution of transmembrane signaling molecule ephrinB2 to ALS pathogenesis, in particular its role in driving motor neuron damage by spinal cord astrocytes. In symptomatic SOD1G93A mice (a well-established ALS model), ephrinB2 expression was dramatically increased in ventral horn astrocytes. Reducing ephrinB2 in the cervical spinal cord ventral horn via viral-mediated shRNA delivery reduced motor neuron loss and preserved respiratory function by maintaining phrenic motor neuron innervation of diaphragm. EphrinB2 expression was also elevated in human ALS spinal cord. These findings implicate ephrinB2 upregulation as both a transcellular signaling mechanism in mutant SOD1-associated ALS and a promising therapeutic target.

Screens in aging-relevant human ALS-motor neurons identify MAP4Ks as therapeutic targets for the disease

Effective therapeutics is much needed for amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease mainly affecting motor neurons. By screening chemical compounds in human patient-derived and aging-relevant motor neurons, we identify a neuroprotective compound and show that MAP4Ks may serve as therapeutic targets for treating ALS. The lead compound broadly improves survival and function of motor neurons directly converted from human ALS patients. Mechanistically, it works as an inhibitor of MAP4Ks, regulates the MAP4Ks-HDAC6-TUBA4A-RANGAP1 pathway, and normalizes subcellular distribution of RANGAP1 and TDP-43. Finally, in an ALS mouse model we show that inhibiting MAP4Ks preserves motor neurons and significantly extends animal lifespan.

Elevated peripheral levels of receptor-interacting protein kinase 1 (RIPK1) and IL-8 as biomarkers of human amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating fatal neurodegenerative disease with no cure. Receptor-interacting protein kinase 1 (RIPK1) has been proposed to mediate pathogenesis of ALS. Primidone has been identified as an old drug that can also inhibit RIPK1 kinase. We conducted a drug-repurposing biomarker study of primidone as a RIPK1 inhibitor using SOD1G93A mice and ALS patients. SOD1G93A mice treated with primidone showed significant delay of symptomatic onset and improved motor performance. One-hundred-sixty-two ALS participants dosed daily with primidone (62.5 mg) completed 24-week follow-up. A significant reduction was showed in serum levels of RIPK1 and IL-8, which were significantly higher in ALS patients than that of healthy controls (P < 0.0001). Serum RIPK1 levels were correlated positively with the severity of bulbar symptoms (P < 0.05). Our study suggests that serum levels of RIPK1 and IL-8 in peripheral can be used as clinical biomarkers for the activation of RIPK1 in central nervous system in human ALS patients. Repurposing primidone may provide a promising therapeutic strategy for ALS. The effect of primidone for the treatment of other inflammatory diseases may also be considered, since the activation of RIPK1 has been implicated in mediating a variety of inflammatory diseases including COVID-19-associated cytokine release syndrome (CRS). (ChiCTR2200060149).

Insight into endoplasmic reticulum-mitochondria contacts in human amyotrophic lateral sclerosis.

Insight into endoplasmic reticulum-mitochondria contacts in human amyotrophic lateral sclerosis.

Intranasal neuropeptide Y1 receptor antagonism improves motor deficits in symptomatic SOD1 ALS mice.

Neuropeptide Y (NPY) is a 36 amino acid peptide widely considered to provide neuroprotection in a range of neurodegenerative diseases. In the fatal motor neuron disease amyotrophic lateral sclerosis (ALS), recent evidence supports a link between NPY and ALS disease processes. The goal of this study was to determine the therapeutic potential and role of NPY in ALS, harnessing the brain-targeted intranasal delivery of the peptide, previously utilised to correct motor and cognitive phenotypes in other neurological conditions. To confirm the association with clinical disease characteristics, NPY expression was quantified in post-mortem motor cortex tissue of ALS patients and age-matched controls. The effect of NPY on ALS cortical pathophysiology was investigated using slice electrophysiology and multi-electrode array recordings of SOD1G93A cortical cultures in vitro. The impact of NPY on ALS disease trajectory was investigated by treating SOD1G93A mice intranasally with NPY and selective NPY receptor agonists and antagonists from pre-symptomatic and symptomatic phases of disease. In the human post-mortem ALS motor cortex, we observe a significant increase in NPY expression, which is not present in the somatosensory cortex. In vitro, we demonstrate that NPY can ameliorate ALS hyperexcitability, while brain-targeted nasal delivery of NPY and a selective NPY Y1 receptor antagonist modified survival and motor deficits specifically within the symptomatic phase of the disease in the ALS SOD1G93A mouse. Taken together, these findings highlight the capacity for non-invasive brain-targeted interventions in ALS and support antagonism of NPY Y1Rs as a novel strategy to improve ALS motor function.

Skeletal muscle as a molecular and cellular biomarker of disease progression in amyotrophic lateral sclerosis: a narrative review.

Amyotrophic lateral sclerosis is a fatal multisystemic neurodegenerative disease with motor neurons being a primary target. Although progressive weakness is a hallmark feature of amyotrophic lateral sclerosis, there is considerable heterogeneity, including clinical presentation, progression, and the underlying triggers for disease initiation. Based on longitudinal studies with families harboring amyotrophic lateral sclerosis-associated gene mutations, it has become apparent that overt disease is preceded by a prodromal phase, possibly in years, where compensatory mechanisms delay symptom onset. Since 85-90% of amyotrophic lateral sclerosis is sporadic, there is a strong need for identifying biomarkers that can detect this prodromal phase as motor neurons have limited capacity for regeneration. Current Food and Drug Administration-approved therapies work by slowing the degenerative process and are most effective early in the disease. Skeletal muscle, including the neuromuscular junction, manifests abnormalities at the earliest stages of the disease, before motor neuron loss, making it a promising source for identifying biomarkers of the prodromal phase. The accessibility of muscle through biopsy provides a lens into the distal motor system at earlier stages and in real time. The advent of "omics" technology has led to the identification of numerous dysregulated molecules in amyotrophic lateral sclerosis muscle, ranging from coding and non-coding RNAs to proteins and metabolites. This technology has opened the door for identifying biomarkers of disease activity and providing insight into disease mechanisms. A major challenge is correlating the myriad of dysregulated molecules with clinical or histological progression and understanding their relevance to presymptomatic phases of disease. There are two major goals of this review. The first is to summarize some of the biomarkers identified in human amyotrophic lateral sclerosis muscle that have a clinicopathological correlation with disease activity, evidence of a similar dysregulation in the SOD1G93A mouse during presymptomatic stages, and evidence of progressive change during disease progression. The second goal is to review the molecular pathways these biomarkers reflect and their potential role in mitigating or promoting disease progression, and as such, their potential as therapeutic targets in amyotrophic lateral sclerosis.

Mature iPSC-derived astrocytes of an ALS/FTD patient carrying the TDP43A90V mutation display a mild reactive state and release polyP toxic to motoneurons.

Astrocytes play a critical role in the maintenance of a healthy central nervous system and astrocyte dysfunction has been implicated in various neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). There is compelling evidence that mouse and human ALS and ALS/FTD astrocytes can reduce the number of healthy wild-type motoneurons (MNs) in co-cultures or after treatment with astrocyte conditioned media (ACM), independently of their genotype. A growing number of studies have shown that soluble toxic factor(s) in the ACM cause non-cell autonomous MN death, including our recent identification of inorganic polyphosphate (polyP) that is excessively released from mouse primary astrocytes (SOD1, TARDBP, and C9ORF72) and human induced pluripotent stem cells (iPSC)-derived astrocytes (TARDBP) to kill MNs. However, others have reported that astrocytes carrying mutant TDP43 do not produce detectable MN toxicity. This controversy is likely to arise from the findings that human iPSC-derived astrocytes exhibit a rather immature and/or reactive phenotype in a number of studies. Here, we have succeeded in generating a highly homogenous population of functional quiescent mature astrocytes from control subject iPSCs. Using identical conditions, we also generated mature astrocytes from an ALS/FTD patient carrying the TDP43A90V mutation. These mutant TDP43 patient-derived astrocytes exhibit key pathological hallmarks, including enhanced cytoplasmic TDP-43 and polyP levels. Additionally, mutant TDP43 astrocytes displayed a mild reactive signature and an aberrant function as they were unable to promote synaptogenesis of hippocampal neurons. The polyP-dependent neurotoxic nature of the TDP43A90V mutation was further confirmed as neutralization of polyP in ACM derived from mutant TDP43 astrocytes prevented MN death. Our results establish that human astrocytes carrying the TDP43A90V mutation exhibit a cell-autonomous pathological signature, hence providing an experimental model to decipher the molecular mechanisms underlying the generation of the neurotoxic phenotype.

Patterns of synaptic loss in human amyotrophic lateral sclerosis spinal cord: a clinicopathological study

Amyotrophic Lateral Sclerosis (ALS) is mainly characterized by the degeneration of corticospinal neurons and spinal α-motoneurons; vulnerable cells display prominent pTDP-43 inclusions. Evidence gathered from genetics, murine models, and iPSC-derived neurons point to the early involvement of synapses in the disease course and their crucial role in the pathogenic cascade. However, pathology studies, with specimens from large post-mortem cohorts, mapping the pattern of synaptic disturbances over clinical and neuropathological hallmarks of disease progression, are currently not available. Thus, the appearance and progression of synaptic degeneration in human ALS patients are currently not known, preventing a full validation of the murine and in vitro models. Here, we investigated the loss of synaptophysin-positive terminals in cervical, thoracic, and lumbar spinal cord samples from a retrospective cohort of n = 33 ALS patients and n = 8 healthy controls, and we correlated the loss of synapses against clinicodemographic features and neuropathological ALS stage. We found that, although dorsal and intermediate spinal cord laminae do not lose synapses, ALS patients displayed a substantial but variable loss of synapses in the ventral horn of lumbar and cervical spinal cord. The amount of synaptic loss was predicted by disease duration, by the clinical site of onset, and by the loss of α-motoneurons, although not by the fraction of pTDP-43-immunopositive α-motoneurons. Taken together, our findings validate the synaptic pathology observed in other models and suggest that pathogenic pathways unfolding in the spinal microenvironment are critical to the progressive disassembly of local synaptic connectivity.

Glycogen accumulation modulates life span in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord. Glial cells, including astrocytes and microglia, have been shown to contribute to neurodegeneration in ALS, and metabolic dysfunction plays an important role in the progression of the disease. Glycogen is a soluble polymer of glucose found at low levels in the central nervous system that plays an important role in memory formation, synaptic plasticity, and the prevention of seizures. However, its accumulation in astrocytes and/or neurons is associated with pathological conditions and aging. Importantly, glycogen accumulation has been reported in the spinal cord of human ALS patients and mouse models. In the present work, using the SOD1G93A mouse model of ALS, we show that glycogen accumulates in the spinal cord and brainstem during symptomatic and end stages of the disease and that the accumulated glycogen is associated with reactive astrocytes. To study the contribution of glycogen to ALS progression, we generated SOD1G93A mice with reduced glycogen synthesis (SOD1G93A GShet mice). SOD1G93A GShet mice had a significantly longer life span than SOD1G93A mice and showed lower levels of the astrocytic pro-inflammatory cytokine Cxcl10, suggesting that the accumulation of glycogen is associated with an inflammatory response. Supporting this, inducing an increase in glycogen synthesis reduced life span in SOD1G93A mice. Altogether, these results suggest that glycogen in reactive astrocytes contributes to neurotoxicity and disease progression in ALS.

Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection.

Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNβ), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNβ than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNβ overproduction during optineurin deficiency both in vitro and in vivo. To investigate the mechanism of IFNβ overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. IFNβ overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNβ overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNβ overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNβ overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.

Intrinsic structural vulnerability in the hydrophobic core induces species-specific aggregation of canine SOD1 with degenerative myelopathy–linked E40K mutation

Canine degenerative myelopathy (DM), a fatal neurodegenerative disease in dogs, shares clinical and genetic features with amyotrophic lateral sclerosis (ALS), a human motor neuron disease. Mutations in the SOD1 gene encoding Cu/Zn superoxide dismutase (SOD1) cause canine DM and a subset of inherited human ALS. The most frequent DM causative mutation is homozygous E40K mutation which induces the aggregation of canine SOD1 but not of human SOD1. However, the mechanism through which canine E40K mutation induces species-specific aggregation of SOD1 remains unknown. By screening human/canine chimeric SOD1s, we identified that the humanized mutation of the 117th residue (M117L), encoded by exon 4, significantly reduced aggregation propensity of canine SOD1E40K. Conversely, introducing a mutation of leucine 117 to methionine, a residue homologous to canine, promoted E40K-dependent aggregation in human SOD1. M117L mutation improved protein stability and reduced cytotoxicity of canine SOD1E40K. Furthermore, crystal structural analysis of canine SOD1 proteins revealed that M117L increased the packing within the hydrophobic core of the β-barrel structure, contributing to the increased protein stability. Our findings indicate that the structural vulnerability derived intrinsically from Met 117 in the hydrophobic core of the β-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.

Developing Targeted Optogenetic Stimulation Approaches to Preserve Upper Airway Function in Amyotrophic Lateral Sclerosis (ALS) Using a Translational Mouse Model

Amyotrophic lateral sclerosis (ALS) results in debilitating upper airway (swallowing and breathing) impairment that profoundly degrades quality of life while increasing the risk of death by almost eight-fold. The underlying cause has been attributed to degeneration of the hypoglossal lower motor neurons (XII LMNs) that control tongue movement; therefore, treatment efforts are focused on slowing XII LMN degeneration. However, there is growing evidence that ALS is not a pure motor neuron disease – it also affects sensory neurons, including those in the nucleus tractus solitarius (NTS) involved in autonomic regulation of swallowing and breathing. We propose that upper airway impairment in ALS may be more effectively treated using sensorimotor approaches that collectively target XII LMNs and the NTS. Here, we are leveraging a translational mouse model of ALS (low copy number SOD1-G93A; LCN-SOD1) that we have shown develops progressive tongue atrophy, XII LMN degeneration, and “motor” dysphagia (slower tongue motility and swallow rates) resembling human ALS. These mice also show clinicopathological evidence of “sensory” dysphagia, specifically a higher swallow threshold (assessed via electrical stimulation of superior laryngeal nerve afferents) and degeneration of the NTS. Using this model, we have been developing tools and procedures to investigate the therapeutic effects of optogenetic stimulation (opto-stim) of XII LMNs and the NTS during voluntary swallow-related behaviors (drinking, eating, grooming) vs. spontaneous breathing in LCN-SOD1 mice. We have already achieved robust expression of the excitatory opsin channelrhodopsin (ChR2) in XII LMNs via adeno-associated virus delivery into the genioglossus muscle or XII nucleus. ChR2 transfection of XII LMNs was well-tolerated, without adverse effects on swallowing/breathing, general health, and survival. Functionally, XII LMN opto-stim evoked robust tongue protrusion (i.e., genioglossus contraction) in anesthetized mice and slowed lick rate during voluntary drinking in awake mice, which we hypothesize is due to resistive force against tongue movement, similar to resistance exercise. Currently, we are exploring the effects of opto-stim (10 ms pulses at 40 or 80 Hz in trains of 1 s ON/OFF for up to 1 h, 2X/wk) as a tongue resistance exercise in pre-clinical LCN-SOD1 mice. In parallel, we are also developing procedures for targeted opsin expression in the NTS via direct injection vs. pharyngeal mucosa application. We hypothesize that NTS opto-stim, in contrast to XII LMN, will assist swallowing by lowering the sensory threshold to unleash higher swallow rates. We propose these novel sensorimotor opto-stim approaches could ultimately translate into minimally invasive targeted treatments for upper airway impairment in ALS patients. This project is funded by NIH R21 NS123761 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Microglial CD68 and L-ferritin upregulation in response to phosphorylated-TDP-43 pathology in the amyotrophic lateral sclerosis brain

Microglia, the innate immune cells of the brain, are activated by damage or disease. In mouse models of amyotrophic lateral sclerosis (ALS), microglia shift from neurotrophic to neurotoxic states with disease progression. It remains unclear how human microglia change relative to the TAR DNA-binding protein 43 (TDP-43) aggregation that occurs in 97% of ALS cases. Here we examine spatial relationships between microglial activation and TDP-43 pathology in brain tissue from people with ALS and from a TDP-43-driven ALS mouse model. Post-mortem human brain tissue from the Neurological Foundation Human Brain Bank was obtained from 10 control and 10 ALS cases in parallel with brain tissue from a bigenic NEFH-tTA/tetO-hTDP-43∆NLS (rNLS) mouse model of ALS at disease onset, early disease, and late disease stages. The spatiotemporal relationship between microglial activation and ALS pathology was determined by investigating microglial functional marker expression in brain regions with low and high TDP-43 burden at end-stage human disease: hippocampus and motor cortex, respectively. Sections were immunohistochemically labelled with a two-round multiplexed antibody panel against; microglial functional markers (L-ferritin, HLA-DR, CD74, CD68, and Iba1), a neuronal marker, an astrocyte marker, and pathological phosphorylated TDP-43 (pTDP-43). Single-cell levels of microglial functional markers were quantified using custom analysis pipelines and mapped to anatomical regions and ALS pathology. We identified a significant increase in microglial Iba1 and CD68 expression in the human ALS motor cortex, with microglial CD68 being significantly correlated with pTDP-43 pathology load. We also identified two subpopulations of microglia enriched in the ALS motor cortex that were defined by high L-ferritin expression. A similar pattern of microglial changes was observed in the rNLS mouse, with an increase first in CD68 and then in L-ferritin expression, with both occurring only after pTDP-43 inclusions were detectable. Our data strongly suggest that microglia are phagocytic at early-stage ALS but transition to a dysfunctional state at end-stage disease, and that these functional states are driven by pTDP-43 aggregation. Overall, these findings enhance our understanding of microglial phenotypes and function in ALS.

Effect of platelet lysate on Schwann-like cell differentiation of equine bone marrow-derived mesenchymal stem cells

Currently, treatment for peripheral nerve injuries in horses primarily relies upon physical therapy and anti-inflammatory drugs. In humans, various treatments using mesenchymal stem cells (MSCs) are being attempted. Therefore, in this study, Schwann-like cell differentiation cultures of equine MSCs were prepared using fetal bovine serum (FBS) and equine platelet lysate (ePL). ePL increased the platelet count to 1 × 106/μl, the optimal concentration for culture. In both groups, an elongated morphology at both ends, characteristic of Schwann cells, was observed under the microscope. Real-time PCR analysis of the expression levels of neuronal markers showed that the ePL group tended to express higher levels of Nestin, Musashi1, and Pax3 than the FBS group. p75 was expressed at low levels in both groups. Immunostaining results showed localization of Nestin in both groups of differentiated cells, but the positive cell rate was significantly higher in the ePL group than in the FBS group. Overall, the ePL gro showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease. This knowledge could be applied translationaly in the treatment of amyotrophic lateral sclerosis in humans.Overall, the ePL group showed good results for Schwann-like cell differentiation, which may be useful for future use in the treatment of equine motor neuron disease and in the treatment of amyotrophic lateral sclerosis in humans.

Nutrition and Equine Performance

Some aspects of energy, protein and vitamin E nutrition of the performance horse are discussed. The amount, dietary source and time of ingestion of energy before exercise can influence performance. In 1989 the National Research Council (NRC) increased their estimates of energy required by racehorses. Recent studies indicate that the increase was reasonable. Many factors, however, can influence energy requirements. Therefore, the best measure would be body weight and composition of the horse. A proper balance of soluble carbohydrate, fiber, fat and protein is essential. Some guidelines are presented. The amount and type energy source given before exercise can influence level of plasma glucose and free fatty acids during exercise, but the effects of these changes in the concentration of metabolites remains to be determined. There is no evidence that increased dietary concentrations of protein are needed and, in fact, may impair performance. Supplemental histidine (to enhance carnosine levels) or carnitine appear to be of limited value for horses fed conventional diets. Dietary concentrations of vitamin E less than the 80 IU/kg recommended by NRC seem to adequately protect against exercise-induced peroxidation. The NRC value may be justified on the basis of immune response, but further studies are needed. Vitamin E has been shown to be involved with familial equine degenerative myeloencephalopathy and may be involved with equine motor neuron disease, a condition considered to be similar to amyotrophic lateral sclerosis in humans.