Severely Stressed Astrocytes Adapt to Injury and Still Protect Neighboring Neurons

Neurodegenerative disorders such as Parkinson\u27s and Alzheimer\u27s disease are projected to affect 15.14 million people by the year 2050. All neurodegenerative diseases are characterized by protein-misfolding stress, or proteotoxicity, in both neurons and glia. However, the impact of proteotoxicity on glia is less well understood than in neurons. Astrocytes are an abundant type of glia and play an essential role in brain homeostasis by supporting neuronal functions. Therefore, one of the major goals of the present study was to identify ways to reduce the impact of proteotoxicity in astrocytes. In Aim 1, we examined natural adaptations to proteotoxic stress in astrocytes. Our studies demonstrate that the astrocytes surviving a severe proteotoxic insult are resistant to subsequent challenges due to upregulation of the antioxidant biomolecule glutathione. Prior to this body of work, only mild stressors were thought to elicit stress tolerance and severe stress was thought to only weaken cells. In contrast with traditional views of reactive astrocytes, severely stressed astrocytes were still able to protect neighboring neurons from proteotoxic injury in our model. In Aim 2, we examined the therapeutic potential of N-acetyl cysteine, a glutathione precursor known to benefit patients with neurological disorders. We discovered that N-acetyl cysteine protected astrocytes from proteotoxic stress independent of glutathione. Instead, the protection afforded by N-acetyl cysteine was abolished by heat shock protein inhibitors, suggesting a novel mechanism of action for N-acetyl cysteine in astrocytes. It is important to promote heat shock protein activity in patients with neurodegenerative conditions because loss of chaperone defenses may contribute to proteotoxicity in the elderly. In Aim 3, we characterized the impact of aging on the expression of several heat shock proteins, many of which are expressed in astrocytes. We discovered age-related changes in multiple heat shock proteins in the substantia nigra, caudoputamen, and olfactory bulb, three regions known to develop Lewy pathology in Parkinson\u27s disease. In conclusion, astrocytes can be protected against protein-misfolding stress by upregulation of endogenous glutathione defenses or by N-acetyl cysteine treatment. Furthermore, some heat shock proteins are increased with aging, perhaps as an endogenous defense against proteotoxic stress in older animals

Astrocytes Do Not Forfeit Their Neuroprotective Roles After Surviving Intense Oxidative Stress

In order to fulfill their evolutionary role as support cells, astrocytes have to tolerate intense oxidative stress under conditions of brain injury and disease. It is well known that astrocytes exposed to mild oxidative stress are preconditioned against subsequent stress exposure in dual hit models. However, it is unclear whether severe oxidative stress leads to stress tolerance, stress exacerbation, or no change in stress resistance in astrocytes. Furthermore, it is not known whether reactive astrocytes surviving intense oxidative stress can still support nearby neurons. The data in this Brief Report suggest that primary cortical astrocytes surviving high concentrations of the oxidative toxicant paraquat are completely resistant against subsequent oxidative challenges of the same intensity. Inhibitors of multiple endogenous defenses (e.g., glutathione, heme oxygenase 1, ERK1/2, Akt) failed to abolish or even reduce their stress resistance. Stress-reactive cortical astrocytes surviving intense oxidative stress still managed to protect primary cortical neurons against subsequent oxidative injuries in neuron/astrocyte co-cultures, even at concentrations of paraquat that otherwise led to more than 80% neuron loss. Although our previous work demonstrated a lack of stress tolerance in primary neurons exposed to dual paraquat hits, here we show that intensely stressed primary neurons can resist a second hit of hydrogen peroxide. These collective findings suggest that stress-reactive astroglia are not necessarily neurotoxic, and that severe oxidative stress does not invariably lead to stress exacerbation in either glia or neurons. Therefore, interference with the natural functions of stress-reactive astrocytes might have the unintended consequence of accelerating neurodegeneration

Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy

Background: Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) disease spectrum, causing both nuclear loss-of-function and cytoplasmic toxic gain-of-function phenotypes. While TDP-43 proteinopathy has been associated with defects in nucleocytoplasmic transport, this process is still poorly understood. Here we study the role of karyopherin-β1 (KPNB1) and other nuclear import receptors in regulating TDP-43 pathology. Methods: We used immunostaining, immunoprecipitation, biochemical and toxicity assays in cell lines, primary neuron and organotypic mouse brain slice cultures, to determine the impact of KPNB1 on the solubility, localization, and toxicity of pathological TDP-43 constructs. Postmortem patient brain and spinal cord tissue was stained to assess KPNB1 colocalization with TDP-43 inclusions. Turbidity assays were employed to study the dissolution and prevention of aggregation of recombinant TDP-43 fibrils in vitro. Fly models of TDP-43 proteinopathy were used to determine the effect of KPNB1 on their neurodegenerative phenotype in vivo. Results: We discovered that several members of the nuclear import receptor protein family can reduce the formation of pathological TDP-43 aggregates. Using KPNB1 as a model, we found that its activity depends on the prion-like C-terminal region of TDP-43, which mediates the co-aggregation with phenylalanine and glycine-rich nucleoporins (FG-Nups) such as Nup62. KPNB1 is recruited into these co-aggregates where it acts as a molecular chaperone that reverses aberrant phase transition of Nup62 and TDP-43. These findings are supported by the discovery that Nup62 and KPNB1 are also sequestered into pathological TDP-43 aggregates in ALS/FTD postmortem CNS tissue, and by the identification of the fly ortholog of KPNB1 as a strong protective modifier in Drosophila models of TDP-43 proteinopathy. Our results show that KPNB1 can rescue all hallmarks of TDP-43 pathology, by restoring its solubility and nuclear localization, and reducing neurodegeneration in cellular and animal models of ALS/FTD. Conclusion: Our findings suggest a novel NLS-independent mechanism where, analogous to its canonical role in dissolving the diffusion barrier formed by FG-Nups in the nuclear pore, KPNB1 is recruited into TDP-43/FG-Nup co-aggregates present in TDP-43 proteinopathies and therapeutically reverses their deleterious phase transition and mislocalization, mitigating neurodegeneration. Graphical Abstract: [Figure not available: see fulltext.]

Heat shock protein defenses in the neocortex and allocortex of the telencephalon

The telencephalic allocortex develops protein inclusions before the neocortex in many age-related proteinopathies. One major defense mechanism against proteinopathic stress is the heat shock protein (Hsp) network. We therefore contrasted Hsp defenses in stressed primary neocortical and allocortical cells. Neocortical neurons were more resistant to the proteasome inhibitor MG132 than neurons from 3allocortical subregions: entorhinal cortex, piriform cortex, and hippocampus. However, allocortical neurons exhibited higher MG132-induced increases in Hsp70 and heat shock cognate 70 (Hsc70). MG132-treated allocortical neurons also exhibited greater levels of protein ubiquitination. Inhibition of Hsp70/Hsc70 activity synergistically exacerbated MG132 toxicity in allocortical neurons more than neocortical neurons, suggesting that the allocortex is more reliant on these Hsp defenses. In contrast, astrocytes harvested from the neocortex or allocortex did not differ in their response to Hsp70/Hsc70 inhibition. Consistent with the idea that chaperones are maximally engaged in allocortical neurons, an increase in Hsp70/Hsc70 activity was protective only in neocortical neurons. Finally, the levels of select Hsps were altered in the neocortex and allocortex invivo with aging

Traumatic injury compromises nucleocytoplasmic transport and leads to TDP-43 pathology

Traumatic brain injury (TBI) is a predisposing factor for many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and chronic traumatic encephalopathy (CTE). Although defects in nucleocytoplasmic transport (NCT) is reported ALS and other neurodegenerative diseases, whether defects in NCT occur in TBI remains unknown. We performed proteomic analysis on Drosophila exposed to repeated TBI and identified resultant alterations in several novel molecular pathways. TBI upregulated nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) proteins as well as alter nucleoporin stability. Traumatic injury disrupted RanGAP1 and NPC protein distribution in flies and a rat model and led to coaggregation of NPC components and TDP-43. In addition, trauma-mediated NCT defects and lethality are rescued by nuclear export inhibitors. Importantly, genetic upregulation of nucleoporins in vivo and in vitro triggered TDP-43 cytoplasmic mislocalization, aggregation, and altered solubility and reduced motor function and lifespan of animals. We also found NUP62 pathology and elevated NUP62 concentrations in postmortem brain tissues of patients with mild or severe CTE as well as co-localization of NUP62 and TDP-43 in CTE. These findings indicate that TBI leads to NCT defects, which potentially mediate the TDP-43 pathology in CTE

Heat shock protein defenses in the neocortex and allocortex of the telencephalon

The telencephalic allocortex develops protein inclusions before the neocortex in many age-related proteinopathies. One major defense mechanism against proteinopathic stress is the heat shock protein (Hsp) network. We therefore contrasted Hsp defenses in stressed primary neo- and allocortical cells. Neocortical neurons were more resistant to the proteasome inhibitor MG132 than neurons from three allocortical subregions: entorhinal cortex, piriform cortex, and hippocampus. However, allocortical neurons exhibited higher MG132-induced increases in Hsp70 and Hsc70. MG132-treated allocortical neurons also exhibited greater levels of protein ubiquitination. Inhibition of Hsp70/Hsc70 activity synergistically exacerbated MG132 toxicity in allocortical neurons more than neocortical neurons, suggesting that the allocortex is more reliant on these Hsp defenses. In contrast, astrocytes harvested from neo- or allocortex did not differ in their response to Hsp70/Hsc70 inhibition. Consistent with the idea that chaperones are maximally engaged in allocortical neurons, an increase in Hsp70/Hsc70 activity was protective only in neocortical neurons. Finally, the levels of select Hsps were altered in neocortex and allocortex in vivo with aging