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

Abstract

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.

Astrocytes Surviving Severe Stress Can Still Protect Neighboring Neurons from Proteotoxic Injury

“…Thus, we tested the hypothesis that loss of Hsp70 activity would lead to loss of astrocytic neuroprotection and applied the Hsp70/Hsc70 inhibitor VER155008 to MG132-treated neuron/astrocyte co-cultures. As shown in our recent work in olfactory bulb and cortical neurons, we found strikingly synergistic effects of inhibitors of proteasomal activity and Hsp70/Hsc70 activity [86, 87]. Remarkably, severely stressed astrocytes protected neurons robustly against this synergistic, profound toxicity (Figure 7).…”

“…For contact co-cultures, primary neocortical neurons were harvested from the primary sensorimotor neocortex of postnatal day 1 Sprague Dawley rats (Charles River) as previously described [63, 64] and plated on top of purified primary cortical astrocytes (described above). Briefly, neocortical tissue was incubated in 10 Units/mL papain (Sigma-Aldrich, Cat.…”

Synergistic stress exacerbation in hippocampal neurons: Evidence favoring the dual‐hit hypothesis of neurodegeneration

“…Heiko Braak observed that the phylogenetically ancient allocortex, of which the hippocampus is a major component, is more vulnerable to tau and alpha‐synuclein pathology in Alzheimer's and Parkinson's disease than the neighboring neocortex (Braak and Braak, ; Braak et al, ). We recently confirmed that neurons from the sensorimotor neocortex are less vulnerable to proteotoxic stress than neurons from three subregions of allocortex: hippocampus, entorhinal cortex, and piriform cortex (Posimo et al, ). Cellular stress from loss of protein homeostasis combined with redox imbalance is a major hallmark of neurodegenerative disorders (Walker et al, ; Jucker and Walker, ; Wang et al, ; Blesa et al, ; Kim et al, ).…”

“…Tissue from the hippocampus was harvested from Sprague Dawley rat brains on postnatal day 1 or 2 (Charles River, Wilmington, MA, USA). Tissue chunks were dissociated and plated as previously described in Neurobasal‐A media with the supplement B27 (Crum et al, ; Posimo et al, ). Cultures were treated with MG132 (EMD Millipore, Billerica, MA, USA) or paraquat (Sigma‐Aldrich, St. Louis, MO, USA) on day in vitro 5 (DIV5) for 24 h. This was referred to as the first hit and was added to the existing media as a 10× solution.…”

“…In order to determine the neuronal purity of the cultures, cells were immunocytochemically labeled for the neuronal marker MAP2 and the astrocyte marker glial fibrillary acidic protein (GFAP) using visible‐range secondary antibodies for higher resolution microscopy, as previously described (Crum et al, ; Posimo et al, ). For the latter experiments, nuclei were stained with Hoechst 33258 (10 μg/mL, bisBenzimide) in phosphate‐buffered saline with 0.3% Triton‐X for 15 min.…”