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Page 8 of 15 Kaya et al. Neuroimmunol Neuroinflammation 2019;6:5 I http://dx.doi.org/10.20517/2347-8659.2018.70

[74]
expression and activation of p53 is the key event in these responses . In the event that irreparable DNA
damage occurs, cells activate their apoptotic machinery, ultimately leading to cell death.

Speedy/RINGO has been shown to prevent p53-dependent apoptosis which is normally induced in response
[12]
to DNA damage in a mitotic human osteosarcoma cell line, U2OS . The survival effect of Speedy/RINGO
on mitotic cells was very strong and significant against a number of extrinsic or intrinsic apoptotic factors
(e.g., carcinogenic-level UV irradiation). Speedy/RINGO helps cells evade apoptosis by inhibiting caspase-3
activation only in the presence of the gene regulatory protein p53 [12,75] .

Speedy/RINGO protein functions as an anti-apoptotic factor in degenerating post-mitotic
neurons
Although there are intrinsic regulatory mechanisms for calcium influx into neurons, a number of insults
such as glutamate neurotoxicity cause deregulation of calcium homeostasis by increasing intraneuronal
calcium influx, as in SCI. This increase in calcium influx induces cystein proteases, including calpain, and
results in pathologic calpain activation. Pathological activation of calpain is known to be one of the most
important neurodegenerative factors triggering apoptosis, which it does by inducing p53 and activating
caspase-3.

Calpain overactivation directly or indirectly induces p53 expression and drives neurons into apoptosis.
Indirectly, overactivated calpain cleaves p35 protein into p25 and p10 fractions. Under normal conditions,
p35 is the partner for non-mitotic neuron-specific kinase cdk5, forming a cdk5/p35 complex. This complex
functions in important cellular events such as neuronal development and maturation [76-78] . When neuronal
2+
calpain is overactivated as a result of increased Ca influx, however, p35 is cleaved by calpain into p25 and
p10 fractions. Like p35, p25 can bind to cdk5, forming a cdk5/p25 complex. However, cdk5/p35 and cdk5/
[79]
p25 complexes differ in both localization and function . The cdk5/p25 complex has been shown to directly
[80]
activate p53, with p53 acting as a substrate for cdk5 . Data indicate that the calcium-mediated calpain
activation observed in SCI results in an increase in p53 expression and activation, leading to caspase-
dependent apoptosis and the resulting degeneration of neurons.

Calpain overactivation leads to increased p53 expression and activation which, in turn, triggers caspase-
[81]
mediated apoptosis of neurons . Apparently, both direct and indirect calpain-induced apoptosis occur in a
p53-dependent fashion.

In addition to its cell cycle regulatory function, Speedy/RINGO has also been shown to function in
preventing apoptosis by inhibiting caspase-3 activation in a p53-dependent manner in mitotic U2OS cells.

Since Speedy/RINGO is primarily a cell cycle regulatory protein, it is highly expressed in mitotic cells
[13]
compared to post-mitotic cells such as neurons . One possible way to prevent p53 mediated apoptosis in
neurons would be to transfect non-mitotic neurons with Speedy/RINGO.

With this in mind, our laboratory designed an in vitro experiment utilizing primary hippocampal neurons
from post-natal (PN0) Sprague-Dawley rats. These neurons were transfected with Speedy/RINGO. After
2+
transfection, calpain was induced using calcium ionophore A23187, facilitating extracellular Ca transport
[13]
into the neurons . Results of our study showed for the first time that Speedy/RINGO, a mitotic cell-specific
protein, is protective against p53-mediated apoptosis in non-mitotic neurons. Calpain induction by A23187
was shown to drive neurons into apoptosis by increasing p53 expression and activating caspase-3, which
is a typical characteristic of caspase-dependent apoptosis. However, overexpression of Speedy/RINGO in
[13]
calpain-induced neurons prevented caspase-3 activation in a p53-dependent manner .

In another recent study, Speedy/RINGO expression levels were shown to substantially decreased in ALS
[14]
motor neurons compared with wild-type controls . As a result of decreased Speedy/RINGO expression,