Fernando E. Santiago - Molecular Biology Research

Fernando E. Santiago, Ph.D.

2015 FAPESP Postdoctoral Fellow

Molecular Biology, with a focus on the Biology of Cancer and Development, and mechanisms of Neurodegeneration

Highlights from Recent Research

...updated December 2015

BAG2 is repressed by NF-kB signaling, and its overexpression is sufficient to shift Ab1-42 from neurotrophic to neurotoxic in undifferentiated SH-SY5Y neuroblastoma. Journal of Molecular Neuroscience (May 2015)

Highlights:

BAG2 expression is lower in undifferentiated cells than in differentiated cells.

Overexpression of BAG2 in undifferentiated cells was sufficient to shift the response to Ab42 from trophic to toxic.

BAG2 expression in undifferentiated cells is negatively regulated by NF-kappaB signaling

Significance:

There remains a significant amount of confusion regarding what actually causes Alzheimer’s disease (AD). On the basis of the histopathological characteristics of AD, plaques (amyloid beta) and tangles (tau), arose the “amyloid hypothesis” and “tau hypothesis” of AD aetiology. The tau hypothesis postulates that dysregulation of tau phosphorylation or a failure to degrade phosphorylated tau (1) interrupts neuronal structure-function via disruption of microtubule dynamics and that (2) phosphorylated forms of tau protein themselves are cytotoxic. This latter point is viewed with some conjecture given the significant amount of contradictory reports in the literature concerning whether tau is actually toxic or not. These apparent contradictions are reviewed by Cowan and Mudher (2013), among others. My personal belief is that tau dysregulation is harmful to neurons, not as a consequence of tau toxicity per se, but as a secondary consequence of tau disruption of processes upon which neurons depend for survival, such as cytoskeleton-dependent intracellular transport, axon maintenance, synapse dynamics and maintenance, and so forth. Neurons appear to behave according to the “use it or lose it” principle and depend upon synaptic activity and neurotrophic factors for survival.

The amyloid hypothesis postulates that extracellular beta-amyloid is harmful to neurons either as a function of its innate toxicity, or as a consequence of its effect upon various neuronal receptors. Notably, treatment of neurons with beta-amyloid has been shown to induce tau phosphorylation. However, none of this addresses two very important observations: (1) beta-amyloid has non-pathological physiological functions and (2) there is an accumulation of beta-amyloid plaques in healthy aging individuals (see references in Santiago et al., 2015). Further, beta-amyloid is toxic in mature neurons, but trophic in undifferentiated neuroblasts.

So what’s the trick?

“Sink or swim, live or die, survive or perish…”

-Daniel Webster, 1826

That BAG2 expression was relatively low in undifferentiated cells—which have a trophic response to Ab42—was our first clue. But BAG-domain family proteins are generally believed to have an anti-apoptotic effect. Despite this, we found that overexpression of BAG2 in undifferentiated cells shifted the response to Ab42 from trophic to toxic. Imagine one gene tipping the scales between life and death.

Like most research, these findings create more questions than they answer. Foremost, while BAG2 is sufficient to induce Ab toxicity in undifferentiated cells, it remains unclear to what degree BAG2 is necessary for Ab toxicity in differentiated cells (or mature, in vivo neurons). If BAG2 function remains consistent in undifferentiated and differentiated cells with regard to Ab42, then these findings may help to explain why beta-amyloid is harmful to mature neurons, and may serve as the mechanistic basis for the apparent duality of beta-amyloid function. In the future, drugs may instead target how a cell responds to beta-amyloid, rather than targeting beta-amyloid itself (Reardon, 2015), as a therapeutic for Alzheimer's disease. Future work will futher characterize the molecular basis for BAG2 activity, as well as the NF-kappaB genes involved in its context-dependent regulation, to shed light on the role BAG2 in deciding the fate of neurons in the central nervous system.

"In the future drugs may target how a cell responds to beta-amyloid,

rather than targeting beta-amyloid itself, as a therapeutic for Alzheimer's disease."

References

Cowan CM, Mudher A. Are tau aggregates toxic or protective in tauopathies? Front Neurol. 2013 Aug 13;4:114. doi: 10.3389/fneur.2013.00114.

Santiago FE, Almeida MC, Carrettiero DC. BAG2 Is Repressed by NF-κB Signaling, and Its Overexpression Is Sufficient to Shift Aβ1-42 from Neurotrophic to Neurotoxic in Undifferentiated SH-SY5Y Neuroblastoma. J Mol Neurosci. 2015 May 19.

Reardon S. Antibody drugs for Alzheimer’s show glimmers of promise. Nature. doi:10.1038/nature.2015.18031

The Co-chaperone BAG2 Mediates Cold-Induced Accumulation of Phosphorylated Tau in SH-SY5Y Cells. Cellular and Molecular Neurobiology (July 2015)

Highlights:

Exposure to cold (34°C) caused a decrease in BAG2 expression in undifferentiated cells, and this decrease in BAG2 expression was accompanied by an increase in phosphorylated tau protein levels

Exposure to cold caused an increase in BAG2 expression in differentiated cells; however this increase in BAG2 expression was not accompanied by a change in phosphorylated tau protein levels

Significance:

I’d be surprised if anyone believed that hypothermia caused a pathological dysregulation of tau per se. However, there are several studies that show that anesthesia-induced hypothermia causes a significant increase in tau phosphorylation. This is interesting because it affords researchers an experimental tool to study the molecular mechanisms that regulate tau phosphorylation, and how this regulation may go awry in the context of pathology.

An earlier publication from Carrettiero and colleagues (2009) showed that BAG2 overexpression resulted in the degradation of phosphorylated tau. Several reports in the literature also observe that phosphorylated tau levels increase on exposure to cold in various model systems (see references in de Paula et al., 2015). Consequently, we sought to determine whether the cold-induced accumulation of phosphorylated tau protein was due to an effect of BAG2.

The results above are interesting to me for several reasons. First, they establish that BAG2 expression is regulated via a temperature-sensitive mechanism. Second, the results demonstrate that the effect of temperature on BAG2 expression is differently regulated in undifferentiated and differentiated cells. Finally, despite an increase in BAG2 expression in cold-exposed differentiated cells, there was no accompanying decrease in phosphorylated tau protein.

This work raises several questions. First, how are we to account for the decrease and increase in BAG2 expression on cold treatment of undifferentiated cells? Perhaps the expression of a co-repressor in undifferentiated cells might shift the activity of a cold-induced transcriptional complex toward inhibition of BAG2. Conversely, a co-activator required for translational activation may be expressed in differentiated, but not in undifferentiated cells.

Second, the relative absence or presence of BAG2 is not necessarily reflected in the levels of phosphorylated tau, as seen in cold-treated differentiated cells. Nor did BAG2 overexpression have an effect on levels of phosphorylated tau in cold-treated differentiated cells. This suggests the possibility that BAG2 requires activation by a cold-inhibited post-translational modification to induce the degradation of phosphorylated tau protein.

Dissection of the cold-sensitive mechanisms responsible for tau phosphorylation may represent a unique opportunity to understand pathways whose dysregulation, perhaps by factors other than temperature, result in the disruption of tau function.

References

Carrettiero DC, Hernandez I, Neveu P, Papagiannakopoulos T, Kosik KS.The cochaperone BAG2 sweeps paired helical filament- insoluble tau from the microtubule. J Neurosci. 2009 Feb 18;29(7):2151-61. doi: 10.1523/JNEUROSCI.4660-08.2009

de Paula CAD*, Santiago FE*, Oliveira ASA, Oliveira FA, Almeida MC, Carrettiero DC. The Co-chaperone BAG2 Mediates Cold-Induced Accumulation of Phosphorylated Tau in SH-SY5Y Cells. Cellular and Molecular Neurobiology. Published – Ahead of Print (July 2015). *Authors contributed equally

BAG2 expression dictates a functional intracellular switch between the p38-dependent effects of nicotine on tau phosphorylation levels via the α7 nicotinic receptor. Experimental Neurology (October 2015)

Highlights:

The effect of nicotine at nicotinic acetylcholine receptors has been reported to be both neuroprotective and neurotoxic, although the molecular basis for this apparently contradictory behavior remains unclear.

The relative expression of BAG2 dictates whether nicotine, via an alpha 7 nicotinic receptor- and p38-mediated mechanism, results in a dose-dependent increase or decrease in tau phosphorylation.

Significance:

The findings presented in this paper are the product of the hard work of two (then) Master’s degree students, Adriele S. A. de Oliveira (photo above; middle) and Laiz Furlan Balioni (top). Immunofluorescence and molecular biology data were replicated in hippocampal primary culture and from these findings an interesting picture began to come into focus.

For years, researchers have reported contradictory findings regarding the effects of nicotine on cultured neurons. In some instances nicotine exerts a neuroprotective effect, rendering a neuron refractory to insult from beta amyloid and oxidative stress, among others; in other contexts, nicotine is neurotoxic (Ferrea and Winterer, 2009). This apparent duality of function is also true of the effects of nicotine on tau phosphorylation – at times it enhances tau phosphorylation, at time is inhibits it.

Because BAG2 has previously been reported to degrade phosphorylated tau protein (Carrettiero et al., 2009), we sought to determine whether BAG2 had any significant input into the nicotine-induced accumulation of phosphorylated tau. We initially expected to see a mere recapitulation of the previously-reported function of BAG2, namely an increase in phospho-tau clearance. What we observed was much more striking, namely that the intracellular abundance of BAG2 protein constituted a molecular “switch” that was capable of diametrically shifting the cellular interpretation of extracellular nicotine from one that encouraged the accumulation of phospho-tau, to one that encouraged a reduction in phospho-tau levels.

"We expected to see a mere recapitulation of reviously-reported BAG2 function.

What we observed was much more striking."

This finding is exciting because it suggests that BAG2 is capable of altering the cellular response to extracellular signaling, in a manner similar to that which I’d previously reported for beta amyloid (Santiago et al., 2015). Work is currently underway in our lab to tease out the precise manner in which BAG2, in essence a molecular “logic gate,” is capable of altering the intracellular landscape to fine-tune cell signaling outcomes.

References

Ferrea S and Winterer G. Neuroprotective and neurotoxic effects of nicotine. Pharmacopsychiatry. 2009 Nov;42(6):255-65. doi: 10.1055/s-0029-1224138.

Carrettiero DC, Hernandez I, Neveu P, Papagiannakopoulos T, Kosik KS. The cochaperone BAG2 sweeps paired helical filament- insoluble tau from the microtubule. J Neurosci. 2009 Feb 18;29(7):2151-61. doi: 10.1523/JNEUROSCI.4660-08.2009.

Santiago FE, Almeida MC, Carrettiero DC. BAG2 Is Repressed by NF-κB Signaling, and Its Overexpression Is Sufficient to Shift Aβ1-42 from Neurotrophic to Neurotoxic in Undifferentiated SH-SY5Y Neuroblastoma. J Mol Neurosci. 2015 Sep;57(1):83-9. doi: 10.1007/s12031-015-0579-5.