Long-Term Cultures of Spinal Cord Interneurons.

Spinal cord interneurons (SpINs) are highly diverse population of neurons that play a significant role in circuit reorganization and spontaneous recovery after spinal cord injury. Regeneration of SpIN axons across rodent spinal injuries has been demonstrated after modification of the environment and neurotrophin treatment, but development of methods to enhance the intrinsic regenerative ability of SpINs is needed. There is a lack of described in vitro models of spinal cord neurons in which to develop new regeneration treatments. For this reason, we developed a new model of mouse primary spinal cord neuronal culture in which to analyze maturation, morphology, physiology, connectivity and regeneration of identified interneurons. Isolated from E14 mice, the neurons mature over 15 days in vitro, demonstrated by expression of maturity markers, electrophysiological patch-clamp recordings, and formation of synapses. The neurons express markers of SpINs, including Tlx3, Lmx1b, Lbx1, Chx10, and Pax2. The neurons demonstrate distinct morphologies and some form perineuronal nets in long-term cultivation. Live neurons in various maturation stages were axotomized, using a 900 nm multiphoton laser and their fate was observed overnight. The percentage of axons that regenerated declined with neuronal maturity. This model of SpINs will be a valuable tool in future regenerative, developmental, and functional studies alongside existing models using cortical or hippocampal neurons

Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats.

A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) treatment is stem cell therapy. Neural progenitors derived from induced pluripotent cells (NP-iPS) might rescue or replace dying motoneurons (MNs). However, the mechanisms responsible for the beneficial effect are not fully understood. The aim here was to investigate the mechanism by studying the effect of intraspinally injected NP-iPS into asymptomatic and early symptomatic superoxide dismutase (SOD)1G93A transgenic rats. Prior to transplantation, NP-iPS were characterized in vitro for their ability to differentiate into a neuronal phenotype. Motor functions were tested in all animals, and the tissue was analyzed by immunohistochemistry, qPCR, and Western blot. NP-iPS transplantation significantly preserved MNs, slowed disease progression, and extended the survival of all treated animals. The dysregulation of spinal chondroitin sulfate proteoglycans was observed in SOD1G93A rats at the terminal stage. NP-iPS application led to normalized host genes expression (versican, has-1, tenascin-R, ngf, igf-1, bdnf, bax, bcl-2, and casp-3) and the protection of perineuronal nets around the preserved MNs. In the host spinal cord, transplanted cells remained as progenitors, many in contact with MNs, but they did not differentiate. The findings suggest that NP-iPS demonstrate neuroprotective properties by regulating local gene expression and regulate plasticity by modulating the central nervous system (CNS) extracellular matrix such as perineuronal nets (PNNs)

A view of the genetic and proteomic profile of extracellular matrix molecules in aging and stroke

IntroductionModification of the extracellular matrix (ECM) is one of the major processes in the pathology of brain damage following an ischemic stroke. However, our understanding of how age-related ECM alterations may affect stroke pathophysiology and its outcome is still very limited.MethodsWe conducted an ECM-targeted re-analysis of our previously obtained RNA-Seq dataset of aging, ischemic stroke and their interactions in young adult (3-month-old) and aged (18-month-old) mice. The permanent middle cerebral artery occlusion (pMCAo) in rodents was used as a model of ischemic stroke. Altogether 56 genes of interest were chosen for this study.ResultsWe identified an increased activation of the genes encoding proteins related to ECM degradation, such as matrix metalloproteinases (MMPs), proteases of a disintegrin and metalloproteinase with the thrombospondin motifs (ADAMTS) family and molecules that regulate their activity, tissue inhibitors of metalloproteinases (TIMPs). Moreover, significant upregulation was also detected in the mRNA of other ECM molecules, such as proteoglycans, syndecans and link proteins. Notably, we identified 8 genes where this upregulation was enhanced in aged mice in comparison with the young ones. Ischemia evoked a significant downregulation in only 6 of our genes of interest, including those encoding proteins associated with the protective function of ECM molecules (e.g., brevican, Hapln4, Sparcl1); downregulation in brevican was more prominent in aged mice. The study was expanded by proteome analysis, where we observed an ischemia-induced overexpression in three proteins, which are associated with neuroinflammation (fibronectin and vitronectin) and neurodegeneration (link protein Hapln2). In fibronectin and Hapln2, this overexpression was more pronounced in aged post-ischemic animals.ConclusionBased on these results, we can conclude that the ratio between the protecting and degrading mechanisms in the aged brain is shifted toward degradation and contributes to the aged tissues’ increased sensitivity to ischemic insults. Altogether, our data provide fresh perspectives on the processes underlying ischemic injury in the aging brain and serve as a freely accessible resource for upcoming research

On the Common Journey of Neural Cells through Ischemic Brain Injury and Alzheimer’s Disease

Ischemic brain injury and Alzheimer’s disease (AD) both lead to cell death in the central nervous system (CNS) and thus negatively affect particularly the elderly population. Due to the lack of a definitive cure for brain ischemia and AD, it is advisable to carefully study, compare, and contrast the mechanisms that trigger, and are involved in, both neuropathologies. A deeper understanding of these mechanisms may help ameliorate, or even prevent, the destructive effects of neurodegenerative disorders. In this review, we deal with ischemic damage and AD, with the main emphasis on the common properties of these CNS disorders. Importantly, we discuss the Wnt signaling pathway as a significant factor in the cell fate determination and cell survival in the diseased adult CNS. Finally, we summarize the interesting findings that may improve or complement the current sparse and insufficient treatments for brain ischemia and AD, and we delineate prospective directions in regenerative medicine

Aryl Hydrocarbon Receptor (AhR)-Mediated Signaling in iPSC-Derived Human Motor Neurons

Exposure to environmental pollutants and endogenous metabolites that induce aryl hydrocarbon receptor (AhR) expression has been suggested to affect cognitive development and, particularly in boys, also motor function. As current knowledge is based on epidemiological and animal studies, in vitro models are needed to better understand the effects of these compounds in the human nervous system at the molecular level. Here, we investigated expression of AhR pathway components and how they are regulated by AhR ligands in human motor neurons. Motor neurons generated from human induced pluripotent stem cells (hiPSCs) were characterized at the molecular level and by electrophysiology. mRNA levels of AhR target genes, CYP1A1 and CYP1B1 (cytochromes P450 1A1/1B1), and AhR signaling components were monitored in hiPSCs and in differentiated neurons following treatment with AhR ligands, 2,3,7,8,-tetrachlodibenzo-p-dioxin (TCDD), L-kynurenine (L-Kyn), and kynurenic acid (KA), by RT-qPCR. Changes in AhR cellular localization and CYP1A1 activity in neurons treated with AhR ligands were also assessed. The neurons we generated express motor neuron-specific markers and are functional. Transcript levels of CYP1B1, AhR nuclear translocators (ARNT1 and ARNT2) and the AhR repressor (AhRR) change with neuronal differentiation, being significantly higher in neurons than hiPSCs. In contrast, CYP1A1 and AhR transcript levels are slightly lower in neurons than in hiPSCs. The response to TCDD treatment differs in hiPSCs and neurons, with only the latter showing significant CYP1A1 up-regulation. In contrast, TCDD slightly up-regulates CYP1B1 mRNA in hiPSCs, but downregulates it in neurons. Comparison of the effects of different AhR ligands on AhR and some of its target genes in neurons shows that L-Kyn and KA, but not TCDD, regulate AhR expression and differently affect CYP1A1 and CYP1B1 expression. Finally, although TCDD does not significantly affect AhR transcript levels, it induces AhR protein translocation to the nucleus and increases CYP1A1 activity. This is in contrast to L-Kyn and KA, which either do not affect or reduce, respectively, CYP1A1 activity. Expression of components of the AhR signaling pathway are regulated with neuronal differentiation and are differently affected by TCDD, suggesting that pluripotent stem cells might be less sensitive to this toxin than neurons. Crucially, AhR signaling is affected differently by TCDD and other AhR ligands in human motor neurons, suggesting that they can provide a valuable tool for assessing the impact of environmental pollutants

Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

<div><p>Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K⁺ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K⁺ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes <i>in situ</i> evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) <i>in situ</i> and <i>in vivo</i> in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K⁺. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K⁺, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K⁺.</p></div

The effect of hypotonic stress or elevated K⁺ on the ECS volume <i>in situ</i>.

<p>The left side: The control values of all experiments were set to 100%, and the relative changes of the values of the extracellular volume fraction α were calculated at 5 min intervals during a 30 min application and a subsequent 60 min washout of mild (<b>A</b>) or severe (<b>B</b>) hypotonic stress or 10 mM K⁺ (<b>C</b>). Each data point represents mean ± S.E.M. The right side: volume regulation during washout at 20 min intervals is expressed as changes in the values reached in the 30^th minute of application, set as 0%. Asterisks indicate significant (*, p<0.05) and very significant (**, p<0.01) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p

Astrocytic volume changes in the cortex of GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice during hypoosmotic stress.

<p>(<b>A, B</b>) Superimposed confocal images of EGFP-labeled cortical astrocytes from GFAP/EGFP/α-Syn^−/− mice obtained in aCSF (control), during a 30-minute exposure to hypoosmotic stress of 250 mOsm/kg (aCSF_H-50; <b>A</b>) or 205 mOsm/kg (aCSF_H-100; <b>B</b>) and during a 60-minute washout. The astrocytic volume was quantified every 10 minutes during exposure to hypoosmotic stress and every 20 minutes during washout. (<b>C, D</b>) Time-dependent changes in the total astrocytic volume in GFAP/EGFP (red) and GFAP/EGFP/α-Syn^−/− mice (green) during a 30-minute exposure to aCSF_H-50 (<b>C left</b>) or to aCSF_H-100 (<b>D left</b>) and during a 60-minute washout. The changes of astrocytic volume during washout were evaluated in each individual cell and expressed as an average percent of cell volume increase/decrease related to the maximal volume after 30 minutes of aCSF_H-50 (<b>C right</b>) or aCSF_H-100 (<b>D right</b>) exposure; these values were set as 0%. Note that α-syntrophin deletion results in smaller astrocyte swelling only during aCSF_H-100 application and in smaller volume changes following mild hypoosmotic stress. Asterisks indicate significant (*, p<0.05), very significant (**, p<0.01) and extremely significant (***, p<0.001) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p

Characterization of double transgenic GFAP/EGFP/α-Syn^−/− mice.

<p>(<b>A</b>) PCR of tail genomic DNA isolated from α-Syn^−/− mice, GFAP/EGFP mice, or double transgenic GFAP/EGFP/α-Syn^−/− mice. Mice lacking the <i>Snta1</i> gene coding α-syntrophin also express <i>Neo</i>, the gene of neomycin resistance, inserted in the gene construct as a positive selectable marker during homologous recombination (top). Western blot analysis detecting α-syntrophin in cortical tissue isolated from α-Syn^−/− mice, GFAP/EGFP mice, or GFAP/EGFP/α-Syn^−/− mice (bottom). (<b>B</b>) Immunohistochemical staining for aquaporin 4 (AQP4) in cortical slices isolated from α-Syn^−/− mice, (<b>C</b>) GFAP/EGFP mice or GFAP/EGFP/α-Syn^−/− mice. Note that α-syntrophin deletion is accompanied by the loss of AQP4 expression on the astrocytic membranes contacting blood vessels.</p

Volume changes in the astrocytic soma and processes during hypotonic stress, increased extracellular K⁺ concentration and OGD.

<p>(<b>A–C</b>) Time-dependent changes in the volume of the astrocytic soma (<b>top</b>) and processes (<b>bottom</b>) in GFAP/EGFP (red) and GFAP/EGFP/α-Syn^−/− mice (green) during a 30-minute application of aCSF_H-100 (<b>A</b>), a 20-minute application of aCSF_K+50 (<b>B</b>) or 20-minute OGD (<b>C</b>), followed by a 60- or 40-minute washout. (<b>D–F</b>) The contribution of the astrocytic soma and processes to the total astrocyte volume changes was expressed as a ratio of the volume changes of both compartments (V_processes/V_soma) after 30 minutes of hypotonic stress and a subsequent 60-minute washout (<b>D</b>), after a 20-minute exposure to aCSF_K+50 and a subsequent 40-minute washout (<b>E</b>), and after 20 minutes of OGD and a subsequent 40-minute washout (<b>F</b>). Note that in GFAP/EGFP mice the swelling of the astrocytic processes prevails (V_processes/V_soma = ∼1.2), while in GFAP/EGFP/α-Syn^−/− the ratio declines towards 1, indicating that the astrocytic processes swell less and the contribution of the cell soma to total astrocyte volume increases. The time-points at which the ratio was calculated are indicated by arrows in A–C. Asterisks indicate significant (*, p<0.05), very significant (**, p<0.01) and extremely significant (***, p<0.001) differences between GFAP/EGFP and GFAP/EGFP/α-Syn^−/− mice.</p