Researchers at Oregon State University have made an important advance toward understanding why certain cells in the nervous system are prone to breaking down and dying, which is what happens in patients with ALS and other neurodegenerative disorders.
The study into the role a protein known as heat shock protein 90 plays in intracellular signaling is a key step on the way to figuring out the reason some motor neurons in the spinal cord die and some do not.
Findings, which could eventually lead to therapies to counter motor neuron death, were published in Experimental Biology and Medicine.
Neurons are cells in the nervous system that carry information to muscles, glands and other nerves.
Motor neurons are large neurons in the spine and brain stem, with long axons extending outside the nervous system to contact muscles and control their movements via contraction.
Researchers led by Alvaro Estevez and Maria Clara Franco of the OSU College of Science have shown that a ubiquitous “protein chaperone,” heat shock protein 90, is particularly sensitive to inhibition in motor neurons that depend for survival on “trophic factors” – small proteins that serve as helper molecules.
Trophic factors attach to docking sites on the surface of nerve cells, setting in motion processes that help keep a cell alive. Research in animal models has shown trophic factors may have the ability to salvage dying neurons.
“It is well known that there are some motor neuron subpopulations resistant to degeneration in ALS, and other subpopulations that are highly susceptible to degeneration,” said Estevez, associate professor of biochemistry and biophysics and the corresponding author on this research.
“Understanding the mechanisms involved in these different predispositions could provide new insight into how ALS progresses and open new alternatives for the development of novel treatments for the disease.”
In this study, a motor-neuron-specific pool of heat shock protein 90, also known as Hsp90, repressed activation of a key cellular receptor and thus was shown to be critical to neuron survival; when Hsp90 was inhibited, motor neuron death was triggered.
The Hsp90 inhibitor used in this research was geldanamycin, an antitumor antibiotic used in chemotherapy.
Findings suggest the drug may have the unintended consequence of decreasing motor neurons’ trophic pathways and thus putting those nerve cells at risk.
“The inhibition of Hsp90 as a therapeutic approach may require the development of inhibitors that are more selective so the cancer cells are targeted and healthy motor neurons are not,” said Franco, assistant professor of biochemistry and biophysics.
ALS, short for amyotrophic lateral sclerosis and also known as Lou Gehrig’s disease, is caused by the deterioration and death of motor neurons in the spinal cord. It is progressive, debilitating and fatal.
ALS was first identified in the late 1800s and gained international recognition in 1939 when it was diagnosed in a mysteriously declining Gehrig, ending the Hall of Fame baseball career of the New York Yankees first baseman.
Known as the Iron Horse for his durability – he hadn’t missed a game in 15 seasons—Gehrig died two years later at age 37.
Peripheral nerve injury induces a robust proregenerative program that drives axon regeneration.
While many regeneration-associated genes are known, the mechanisms by which injury activates them are less well-understood.
To identify such mechanisms, we performed a loss-of-function pharmacological screen in cultured adult mouse sensory neurons for proteins required to activate this program. Well-characterized inhibitors were present as injury signaling was induced but were removed before axon outgrowth to identify molecules that block induction of the program. Of 480 compounds, 35 prevented injury-induced neurite regrowth.
The top hits were inhibitors to heat shock protein 90 (HSP90), a chaperone with no known role in axon injury. HSP90 inhibition blocks injury-induced activation of the proregenerative transcription factor cJun and several regeneration-associated genes.
These phenotypes mimic loss of the proregenerative kinase, dual leucine zipper kinase (DLK), a critical neuronal stress sensor that drives axon degeneration, axon regeneration, and cell death. HSP90 is an atypical chaperone that promotes the stability of signaling molecules. HSP90 and DLK show two hallmarks of HSP90–client relationships: (i) HSP90 binds DLK, and (ii) HSP90 inhibition leads to rapid degradation of existing DLK protein. Moreover, HSP90 is required for DLK stability in vivo, where HSP90 inhibitor reduces DLK protein in the sciatic nerve.
This phenomenon is evolutionarily conserved in Drosophila. Genetic knockdown of Drosophila HSP90, Hsp83, decreases levels of Drosophila DLK, Wallenda, and blocks Wallenda-dependent synaptic terminal overgrowth and injury signaling. Our findings support the hypothesis that HSP90 chaperones DLK and is required for DLK functions, including proregenerative axon injury signaling.
Axon injury occurs in response to trauma, metabolic and toxic insults, and neurodegenerative and genetic diseases. Understanding axonal injury response pathways may lead to strategies for axonal repair.
While mammalian central axon regeneration is stunted by a nonpermissive environment and low intrinsic growth capacity (1, 2), peripheral axons can undergo robust regeneration and thus, provide an attractive system to study proregenerative signaling.
Peripheral nerve injury activates cytoskeletal remodeling that transforms the injured axon tip into a growth cone (1).
Concurrently, local signaling molecules detect the injury and drive retrograde signals to the nucleus to induce expression of regeneration-associated genes (RAGs) (3). This transcriptional program transforms the neuron into a proregenerative state to enable efficient axon regeneration (4, 5).
DLK promotes retrograde transport of injury signals and is required for axon regeneration in mice, Drosophila, and Caenorhabditis elegans (9⇓⇓–12). Along with DLK, a handful of other kinases, transcription factors, and histone modifiers drive regenerative axon signaling, and other factors are likely yet undiscovered (13⇓–15). We sought to identify additional components of the axon injury response, including previously unidentified pathways or undescribed regulators of known signals, such as DLK.
To accomplish this, we developed an in vitro screen to identify injury signals required for induction of the proregenerative program. We took advantage of the preconditioning phenomenon, in which a conditioning injury activates the regeneration program and a second test injury assays its state (16).
Traditionally, this paradigm is performed in vivo, but we and others have recently described an in vitro version of this assay in which dissection of mouse dorsal root ganglia (DRG) neurons serves as the preconditioning lesion (17⇓–19).
Twenty-four hours later, the regeneration program is active, and we administer the testing injury via replating of the neurons. Preconditioned neurons grow extensive neurites in a short time compared with uninjured neurons.
The major advantage that this assay has over the in vivo counterpart is that injury signaling is induced in culture and therefore is amenable to pharmacological perturbations. Importantly, drugs are present only during induction of the regeneration program, not during axon sprouting or outgrowth.
We miniaturized this assay to develop a loss-of-function screening platform to identify small molecules that inhibit induction of the axon regeneration program. From a 480-compound library, we found inhibitors of proteins with no known role in axon injury signaling and inhibitors to several known injury signals.
Our analysis focused on the most potent hits, heat shock protein 90 (HSP90) inhibitors, which blocked many of the molecular components of the proregenerative program and the subsequent promotion of robust neurite outgrowth.
These phenotypes mimic those seen with loss of DLK. Because HSP90 is a chaperone that facilitates the activity of signaling molecules, including kinases, we tested the hypothesis that HSP90 is required for axon injury signaling as a chaperone for DLK (20, 21). In support of this hypothesis, we show that HSP90 binds DLK and is required for the stability of existing DLK protein.
We show that HSP90 regulates DLK levels in vivo in mice and Drosophila. Moreover, we show that HSP90 is required for both DLK-dependent axon injury signaling and developmental synaptic terminal overgrowth in Drosophila. Together, these data demonstrate that DLK is an evolutionarily conserved client of HSP90, that axon injury signaling requires HSP90 activity, and that a primary mechanism by which HSP90 facilitates injury signaling is to chaperone DLK.
HSP90 Inhibition Prevents Activation of the Regeneration Program.
From this group of targets, we chose to perform a more detailed characterization of the chaperone HSP90. Two HSP90 inhibitors, geldanamycin and its less toxic analog 17-N-allylamino-17-demethoxygeldanamycin (17AAG), were hits at both doses, with the high dose of 17AAG being the number one hit in the screen.
Moreover, there is no known role for HSP90 in axon injury signaling or axon regeneration. In the manual replating assay in which the longest neurite per neuron is imaged and quantified by hand, 1 µM 17AAG was sufficient to inhibit the regeneration program over fivefold compared with DMSO-treated controls (Fig. 2 A and B).
To assess whether the block of axon regeneration was due to HSP90 inhibition, we tested a structurally distinct HSP90i, ganetespib (GT), and found that it also blocked preconditioned axon growth.
As a comparison, we inhibited the essential proregenerative kinase, DLK, with a recently characterized potent and selective DLK inhibitor (DLKi), GNE-3511 (32), and we found that it also strongly blocked preconditioned axon regrowth. Although 17AAG did not score as toxic in the 96-well format, before proceeding to mechanistic studies, we performed a more rigorous analysis of toxicity by quantifying cell death. Live cells were defined as both positive for the mitochondrial potential marker, Tetramethylrhodamine, methyl ester (TMRM), and negative for the cell death marker, YoPro. Neurons treated with either DMSO or 17AAG for 24 h displayed ∼25% cell death, an expected percentage, as not all cells survive dissociation and plating (Fig. 2 Cand D).
Those treated with the mitochondrial poison carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were nearly all dead. Lastly, we asked if 17AAG-treated neurons retained the ability to grow neurites long after drug washout to test whether 17AAG permanently abolished the ability of neurons to grow neurites. We performed the replating assay as previously described, but instead of fixing the neurons at 18 h, we fixed at 72 h, allowing ample time for neurons to reactivate their regeneration program and grow long neurites. Indeed, both DMSO-treated and 17AAG-treated neurons grow extensive neurites 72 h after drug washout and replating (Fig. 2E).
Collectively, these data demonstrate that 17AAG blocks functional activation of the regeneration program and is not toxic to adult sensory neurons.
The axon regeneration program promotes axonal outgrowth via induction of a molecular program that includes transcription factor activation, transcriptional induction of RAGs, and the production of axon growth-associated proteins (4). To explore how HSP90i inhibits the regeneration program, we assessed molecular components of the regeneration program. Twenty-four hours after the conditioning injury, instead of replating the neurons and measuring neurite outgrowth, we quantified the levels of phosphorylated (activated) cJun (p-cJun), up-regulation of regeneration-associated proteins superior cervical ganglion 10 (SCG10) and growth-associated protein 43 (GAP43), and transcriptional induction of two RAGs: Small proline-rich protein 1a (Sprr1a) and Galanin. cJun is the transcription factor target of JNK, and it promotes axon regeneration (33). cJun phosphorylation increased approximately fivefold between 1 and 24 h postplating (Fig. 3 A and B). Neurons treated with 17AAG only increased their p-cJun signal 1.6-fold. As a positive control, we tested the effect of DLKi, since DLK is required for cJun phosphorylation after peripheral nerve injury in vivo (8). As expected, application of DLKi blocks the phosphorylation of cJun in this system. SCG10 and GAP43 are injury-induced cytoskeletal remodelers that are commonly used molecular markers of regenerating axons (10, 17). In neurons cultured for 24 h, SCG10 and GAP43 increased approximately 7- (Fig. 3 A and C) and 2.5-fold (Fig. 3 A and D), respectively. Surprisingly, neither 17AAG nor DLKi had a significant effect on the induction of these proteins. Sprr1a and Galanin are injury-induced transcripts that each encode axon growth proteins (33, 34). At 24 h after plating, both Sprr1a and Galanin are robustly up-regulated (Fig. 3E). Neurons treated with 17AAG or DLKi fail to up-regulate these genes in response to axon injury. Hence, inhibition of HSP90 potently suppresses axonal outgrowth after injury while blocking some but not all molecular components of the regeneration program. HSP90 inhibitor is not poisoning the entire regenerative program; instead, it may inhibit specific signaling pathways.
More information: Amy L Strayer et al. Highlight article: Ligand-independent activation of the P2X7 receptor by Hsp90 inhibition stimulates motor neuron apoptosis, Experimental Biology and Medicine(2019). DOI: 10.1177/1535370219853798