Virve Kärkkäinen

Nrf2 Regulates Neurogenesis and Protects Neural Progenitor Cells Against Aβ Toxicity

Neural stem/progenitor cells (NPCs) proliferate and produce new neurons in neurogenic areas throughout the lifetime. While these cells represent potential therapeutic treatment of neurodegenerative diseases, regulation of neurogenesis is not completely understood. We show that deficiency of nuclear factor erythroid 2‐related factor (Nrf2), a transcription factor induced in response to oxidative stress, prevents the ischemia‐induced increase in newborn neurons in the subgranular zone of the dentate gyrus. Consistent with this finding, the growth of NPC neurospheres was increased by lentivirus‐mediated overexpression of Nrf2 gene or by treatment with pyrrolidine dithiocarbamate (PDTC), an Nrf2 activating compound. Also, neuronal differentiation of NPCs was increased by Nrf2 overexpression or PDTC treatment but reduced by Nrf2 deficiency. To investigate the impact of Nrf2 on NPCs in Alzheimers disease (AD), we treated NPCs with amyloid beta (Aβ), a toxic peptide associated with neurodegeneration and cognitive abnormalities in AD. We found that Aβ1–42‐induced toxicity and reduction in neurosphere proliferation were prevented by Nrf2 overexpression, while Nrf2 deficiency enhanced the Aβ1–42‐induced reduction of neuronal differentiation. On the other hand, Aβ1–40 had no effect on neurosphere proliferation in wt NPCs but increased the proliferation of Nrf2 overexpressing neurospheres and reduced it in Nrf2‐deficient neurospheres. These results suggest that Nrf2 is essential for neuronal differentiation of NPCs, regulates injury‐induced neurogenesis and provides protection against Aβ‐induced NPC toxicity. Stem Cells 2014;32:1904–1916

Neurotransmitter responsiveness during early maturation of neural progenitor cells.

Neurotransmitters are potential regulators of proliferation and differentiation of neural progenitor cells (NPC). To gain insight into the dynamics of neurotransmitter responsiveness, neurospheres were prepared from the lateral ventricles of postnatal day 6/7 mice. Individual NPCs migrating out from spheres were simultaneously monitored using Ca(2+) imaging, during the initial 8 days of differentiation, at an area between the inner edge of the sphere and outer periphery of the area of migration. At the first day of differentiation most cells showed metabotropic responses (Ca(2+) discharge from stores) to glutamate (pharmacologically identified as metabotropic glutamate receptor 5, mGluR 5), norepinephrine (NE), acetylcholine (Ach) and ATP, and a smaller proportion of cells also responded to substance P (SP). When outside the neurosphere, many of mGluR5 responding cells gained immunostaining for markers of neuronal lineage (Tuj-1 and NeuN). The number of cells responding through mGluR5 (and responses to Ach, NE and SP) showed during subsequent days of differentiation (day 2-3 onwards) a decline with time and progressively disappeared at the outer periphery of the area of migration. Conversely the number ionotropic glutamate responses as well as responses to depolarization increased in this area. After 5-8 days of differentiation mGluR5 responses could only be observed at the very inner edge of the neurosphere. At 8 days the migrated cells showed very robust ionotropic responses to glutamate, NMDA and depolarization comparable to mature neurons. Taken together, the data presented here suggest that differentiation of NPCs is a dynamic process triggered by cell migration, which leads to a loss of regulatory influences imposed by the inner milieu of the neurosphere. The subsequent switch or loss of metabotropic responses to glutamate, SP, NE, Ach and ATP with the gain of excitable characteristics such as ionotropic responses appears to be a key event in the final differentiation process.

Brain environment and Alzheimer's disease mutations affect the survival, migration and differentiation of neural progenitor cells.

Enhancement of neurogenesis and stem cell transplantation are potential therapies for neurodegenerative diseases. In Alzheimers disease (AD) newborn neural cells and the transplanted cells encounter a diseased brain where the accumulation of toxic amyloid-β (Aβ) peptides disturbs normal functions and interactions of brain cells. In addition, ADlinked mutatations in newborn neurons or autologously transplanted neural progenitor cells (NPCs) are likely to affect the fate of these cells. Here we analyzed the effect of AD-linked APdE9 mutant on NPCs in culture and by isolating NPCs from APdE9 transgenic mice and transplanting them into APdE9 and wild type mouse brain. We show that the brain environment in APdE9 mutant mice reduced astrocytic differentiation but increased the survival and migration of NPCs in vivo. APdE9 mutation of NPCs increased neuronal differentiation also in vitro. Instead extracellular Aβ(42) peptide decreased the survival, neuronal differentiation and migration of NPCs in vitro, whereas Aβ(40) had an opposite effect. NPC transplantation induced brain neurogenesis, which was not altered in Aβ burdened APdE9 brain or by APdE9 mutant in NPCs. Thus, AD-linked mutations in newborn neural cells or NPCs do not compromise the utilization of enhanced neurogenesis or autologous NPC transplantation as potential therapies. The results suggest that combining the treatments resulting in reduced Aβ(42) and enhanced neurogenesis may be one therapeutic approach to be explored in AD.

Metabotropic glutamate receptor 5 responses dictate differentiation of neural progenitors to NMDA-responsive cells in fragile X syndrome

Disrupted metabotropic glutamate receptor 5 (mGluR5) signaling is implicated in many neuropsychiatric disorders, including autism spectrum disorder, found in fragile X syndrome (FXS). Here we report that intracellular calcium responses to the group I mGluR agonist (S)−3,5‐dihydroxyphenylglycine (DHPG) are augmented, and calcium‐dependent mGluR5‐mediated mechanisms alter the differentiation of neural progenitors in neurospheres derived from human induced pluripotent FXS stem cells and the brains of mouse model of FXS. Treatment with the mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl)‐pyridine (MPEP) prevents an abnormal clustering of DHPG‐responsive cells that are responsive to activation of ionotropic receptors in mouse FXS neurospheres. MPEP also corrects morphological defects of differentiated cells and enhanced migration of neuron‐like cells in mouse FXS neurospheres. Unlike in mouse neurospheres, MPEP increases the differentiation of DHPG‐responsive radial glial cells as well as the subpopulation of cells responsive to both DHPG and activation of ionotropic receptors in human neurospheres. However, MPEP normalizes the FXS‐specific increase in the differentiation of cells responsive only to N‐methyl‐d‐aspartate (NMDA) present in human neurospheres. Exposure to MPEP prevents the accumulation of intermediate basal progenitors in embryonic FXS mouse brain suggesting that rescue effects of GluR5 antagonist are progenitor type‐dependent and species‐specific differences of basal progenitors may modify effects of MPEP on the cortical development.

ADAMTS-4 promotes neurodegeneration in a mouse model of amyotrophic lateral sclerosis

BackgroundA disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) proteoglycanases are specialized in the degradation of chondroitin sulfate proteoglycans and participate in mechanisms mediating neuroplasticity. Despite the beneficial effect of ADAMTS-4 on neurorepair after spinal cord injury, the functions of ADAMTS proteoglycanases in other CNS disease states have not been studied. Therefore, we investigated the expression, effects and associated mechanisms of ADAMTS-4 during amyotrophic lateral sclerosis (ALS) in the SOD1G93A mouse model.ResultsADAMTS-4 expression and activity were reduced in the spinal cord of SOD1G93A mice at disease end-stage when compared to WT littermates. To counteract the loss of ADAMTS-4, SOD1G93A and WT mice were treated with saline or a recombinant ADAMTS-4 before symptom onset. Administration of ADAMTS-4 worsened the prognosis of SOD1G93A mice by accelerating clinical signs of neuromuscular dysfunctions. The worsened prognosis of ADAMTS-4-treated SOD1G93A mice was accompanied by increased degradation of perineuronal nets enwrapping motoneurons and increased motoneuron degeneration in the lumbar spinal cord. Motoneurons of ADAMTS-4-treated SOD1G93A mice were more vulnerable to degeneration most likely due to the loss of their extracellular matrix envelopes. The decrease of neurotrophic factor production induced by ADAMTS-4 in vitro and in vivo may also contribute to a hostile environment for motoneuron especially when devoid of a net.ConclusionsThis study suggests that the reduction of ADAMTS-4 activity during the progression of ALS pathology may be an adaptive change to mitigate its neurodegenerative impact in CNS tissues. Therapies compensating the compromized ADAMTS-4 activity are likely not promising approaches for treating ALS.

Loss of CLN5 causes altered neurogenesis in a childhood neurodegenerative disorder

ABSTRACT Neural stem/progenitor cells (NPCs) generate new neurons in the brain throughout an individuals lifetime in an intricate process called neurogenesis. Neurogenic alterations are a common feature of several adult-onset neurodegenerative diseases. The neuronal ceroid lipofuscinoses (NCLs) are the most common group of inherited neurodegenerative diseases that mainly affect children. Pathological features of the NCLs include accumulation of lysosomal storage material, neuroinflammation and neuronal degeneration, yet the exact cause of this group of diseases remains poorly understood. The function of the CLN5 protein, causative of the CLN5 disease form of NCL, is unknown. In the present study, we sought to examine neurogenesis in the neurodegenerative disorder caused by loss of Cln5. Our findings demonstrate a newly identified crucial role for CLN5 in neurogenesis. We report for the first time that neurogenesis is increased in Cln5-deficient mice, which model the childhood neurodegenerative disorder caused by loss of Cln5. Our results demonstrate that, in Cln5 deficiency, proliferation of NPCs is increased, NPC migration is reduced and NPC differentiation towards the neuronal lineage is increased concomitantly with functional alterations in the NPCs. Moreover, the observed impairment in neurogenesis is correlated with increased expression of the pro-inflammatory cytokine IL-1β. A full understanding of the pathological mechanisms that lead to disease and the function of the NCL proteins are critical for designing effective therapeutic approaches for this devastating neurodegenerative disorder. Editors’ choice: This study demonstrates neurogenic alterations in the most common neurodegenerative disease of children and reports a novel function of the CLN5 protein.

The effects of metabotropic glutamate antagonist on neural stem cells in fragile X syndrome

oxidantand inflammatory-mediated diseases, including brain aging. In the present study we report that treatment for 4 months of senescent rats with Acetylcarnitine (LAC) is able to induce heme oxygenase-1, Hsp70 and SOD-2, and that this effect is associated with up-regulation of GSH levels. In addition, by using redox proteomics, we provide a valuable insight into the mechanism of age-related protein oxidation and the effect of LAC in reducing oxidative stress associated with functional impairment in aged brains. Redox proteomics analysis of hippocampus (HP) and cerebral cortex (CX), brain regions in which all indices of oxidative modification are elevated in brain aging showed that the specific carbonyl levels of three proteins, hemoglobin, cofilin 1 and beta-actin, are significantly increased in HP of senescent rats. All specific carbonyl levels of these proteins are reduced by LAC administration in old rats. In the CX of senescent rats, the specific carbonyl levels of eight proteins: heat shock protein 70, glyoxylase 1, beta-actin, 3-mercaptopyruvate sulfurtransferase, peroxiredoxin 1, phosphoribosyl pyrophosphate synthetase 1, and fumarase, were increased. LAC administration was able to reduce the specific carbonyl levels of all these proteins which are involved in three impaired processes in aged brains: antioxidant defence, mitochondrial function and synaptic plasticity. Treatment by LAC may improve the decline of these functions and therefore LAC could be considered as a potential therapeutic agent for treatment of cognitive decline in aging. Therapeutic strategies focussing on acetylcarnitine treatment may represent a crucial mechanism of defence against free radical-induced damage of critical proteins occurring in aging brain.