SUMOtherapeutics for Ischemic Stroke

All this blather on neuroprotection and they never explain that after reperfusion a lot of dying of neurons continues for quite a while. The Rockefeller University in 2008 called it the cascade of death. I call it the neuronal cascade of death  to signify the extreme importance of preventing it from happening, neuroprotection is way too bland a word to signify immediacy at all. I lost maybe 4.4 billion neurons in the first week because my doctors DID NOTHING to stop the neuronal cascade of death in the first week!

It should be called the neuronal cascade of death signifying extreme urgency while neuroprotection means nothing to survivors and doctors use that to bamboozle patients; 'We didn't get neuroprotection to work'.  As compared to the statement; 'We failed at stopping the neuronal cascade of death  thus allowing millions to billions of your neurons to DIE, DIE, DIE!'  

WHICH STATEMENT WILL GET YOUR DOCTORS TO SOLVE STROKE? )

 SUMOtherapeutics for Ischemic Stroke

Paramesh Karandikar1; Jakob V. E. Gerstl, MBBS2; Ari D. Kappel, MD2,3; Sae-Yeon Won, MD4; Daniel Dubinski,
MD4, MSc; Monica Emili Garcia-Segura5,6; Florian A. Gessler, MD, PhD4; Alfred Pokmeng See, MD3; Luca Peruzzotti
Jametti, MD, PhD5,6+; Joshua D. Bernstock, MD, PhD, MPH2, 4, 7, +*
1 T. H. Chan School of Medicine, University of Massachusetts, Worcester, MA, USA
2 Department of Neurosurgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
3 Department of Neurosurgery, Boston Children’s Hospital, Boston, MA, USA
4 Department of Neurosurgery, University Medicine Rostock, Rostock, Germany
5 Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
6 NIHR Biomedical Research Centre, University of Cambridge, Cambridge, UK
7 Koch Institute for Integrated Cancer Research, MIT, Cambridge, MA, USA
* Correspondence: Joshua D. Bernstock MD, PhD, MPH; jbernstock@bwh.harvard.edu
+ These authors contributed equally to this work.

Abstract:  

Small, Ubiquitin-like Modifier (SUMO) is a post translational modifier with a profound
influence on several key biological processes including the mammalian stress response. Of particular interest is its neuroprotective effects, first recognized in the 13-lined ground squirrel (Ictidomys tridecemlineatus), in the context of hibernation torpor. Although the full scope of the SUMO pathway is yet to be elucidated, observations of its importance in managing neuronal responses to ischemia, maintaining ion gradients, and preconditioning of neural stem cells, make it a promising therapeutic target for acute cerebral ischemia. Recent advances in high-throughput screening have enabled the identification of small molecules that can upregulate SUMOylation, some of which have been validated in pertinent preclinical models of cerebral ischemia. Accordingly, the present review aims to summarize current knowledge and highlight the translational potential of the SUMOylation pathway in brain ischemia.
Keywords: stroke; ischemia; neuroprotection; SUMOylation; experimental therapeutics
1. Introduction
Despite landmark developments in chemical thrombolysis and mechanical thrombectomy in the past decades, ischemic stroke remains one of the greatest drivers of global
disease burden [1-4]. Effective management options are often not available due to temporality of presentation and lack of access to specialist medical facilities, even in high income settings. Eligibility for alteplase/tenecteplase is dependent on patient presentation within 4.5-9 hours and absence of disqualifying comorbidities [5]. Endovascular thrombectomy is an effective method for revascularization, but not available to many patients as they are either not candidates for intervention, do not have access to a skilled operator at a stroke capable center, or may not present within the time window for intervention. In fact, a significant fraction of patients with ischemic stroke do not benefit from rapid recanalization—for example, between 2012-2018 in the United States, only 3.5% of stroke patients received thrombectomy, 9.3% received tPA, and 1% received both [6]. While the positive results reported in the DAWN and DEFUSE-3 trials have likely spurred wider utilization of these methods, the fact remains that the treatment paradigm remains the same: existing care for stroke focuses on the physiologic effects of flow and perfusion on bulk tissue and does not account for the cellular or molecular biological mechanisms of ischemic stroke. Even with advances in imaging to identify patients with less tissue injury and persistent at risk tissue, about half of patients taken for stroke intervention still have unfavorable outcomes (54% [7], 56% [2], 51% [3]). On the contrary, RESCUE-Japan LIMIT [8] , SELECT2
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[9], and ANGEL-ASPECT [10] show that patients with significant tissue injury can still
benefit from reperfusion. While structural vessel occlusion is the initiating event in clinical stroke, the pathobiological sequelae that unfolds as a result of such ischemia must ultimately also be targeted and as such the expansion of treatment paradigms to include novel neuroprotetctive
therapies will be required both for patients that are successfully treated (e.g., subjective to
reperfusion injury) and those that are not. Accordingly, herein, we provide a clinically
oriented summary of the current understanding of the role of the Small, Ubiquitin-like
Modifier (SUMO) protein in endogenous neuroprotective mechanisms, recapitulate
known therapeutic candidates acting on SUMO pathways (SUMOtherapeutics), and conclude by exploring potential future directions and approaches within the context of ischemic stroke.
1.1 Biological significance of the SUMO pathway
SUMO is a protein intrinsically involved in orchestrating physiological responses to
hypoxia, hypothermia, and DNA damage [11]. SUMO conjugation of proteins (SUMOy-
lation) was first implicated in the torpor conditioning of the 13-lined ground squirrel (Ic-
tidomys tridecemlineatus). The analogous nature of this physiological mechanism of hibernation to the pathological mechanism of ischemic stroke and arousal to reperfusion has made it a direction for inquiry in the context of neuroprotection [12-14]. Postmortem observation of increased SUMOylation within the penumbra of ischemic stroke in human victims has made the potential exploitation of this pathway of clinical interest [15]. The SUMO pathway exerts its effects by way of post-translation modification akin to ubiquitinylation. However, while only 29 variants of ubiquitin binding domains have been identified in humans at the time of writing, over 14,000 SUMO binding domains have been found in human cells [16]. Conversely, whereas ubiquitinylation involves one of almost three dozen forms of the E2 conjugase, SUMOylation relies solely on one—UBC9[17]. This combination of a vast number of SUMO binding domains coupled with a universal reliance on a single E2 conjugase has significant potential as a wide-reaching yet approachable therapeutic target.
Although 4 SUMO paralogs have been identified in humans, SUMO-1 and SUMO-
2/3 (SUMO-2 and SUMO-3 being almost identical) represent the prevalent isotypes [17,
18]. The act of conjugating/deconjugating SUMO, SUMOylation and de-SUMOylation, is
primarily carried out by seven Sentrin specific proteases known as SENP1-7. Of this family, SENP1 (key for maturing translated SUMO) and SENP2 display the broadest substrate
affinity for SUMO isotypes and have been found preferentially within the nucleus and
nuclear pore complex. The other SENPs are either selectively active on a particular SUMO
isoform or are not involved in SUMO maturation, making them of lesser interest for SU-
MOtherapeutics engineered for global upregulation. Other SUMO-involved enzymes
such as DeSI-1, DeSI-2, and USPL1 have been identified but have been found to have weak
endopeptidase activity and are not thought to be involved in global SUMOylation to the
extent of SENP1 and SENP2 [11, 19, 20].
The exact extent of control exerted by SUMO and the bounds of its physiological in-
terplay remain to be fully elucidated. SUMOylation is involved in numerous processes
spanning most organ systems, with downstream effects occasionally resulting in simultaneous upregulation and downregulation of protein machinery like HIF-1a [11]. Knockout
experiments have demonstrated that SUMO has a role, albeit undefined, in emotion, cog-
nition, anxiety, and episodic memory [21, 22]. Preclinical work has shown that SUMO
serves as a regulator of critical cellular processes including DNA repair, axonal trafficking,
brain development, as well as neuronal plasticity and neurotransmission [19, 20, 23-26].
Furthermore, SUMOylation and de-SUMOylation have been identified as potential driv-
ers of various CNS pathologies. Glioblastomas have been found to overexpress
SUMO[22]; increased SUMOylation has been associated with Alzheimer’s Disease and
Huntington’s Disease; and both increased SUMO1ylation and decreased SUMO2/3ylation
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 6 April 2023 doi:10.20944/preprints202304.0094.v1

have been implicated with alpha-synuclein aggregation in Parkinson’s disease [27]. More-
over, SUMOylation is known to play a role in cardiac [28, 29], renal [30], pulmonary, he-
patic and mesenteric ischemic disease processes [14]. These observations, coupled with
reports on the critical role of SUMO in cell survival during hyperacute ischemic pathologies such as myocardial infarction[31] make it of particular interest for the management
of acute ischemic stroke.

1.2 Role of SUMO in neuroprotection

The neuroprotective effects of SUMOylation have been widely observed both in vitro

and in vivo. Due to the role of SUMO as the “modifier of modifiers” and the great physio-
logical distance between the act of SUMOylation and its ultimate effect; a comprehensive
pattern of association between end-effects and neuroprotection remains to be clearly
demonstrated. The importance of SUMO in preserving neuronal integrity first observed
in natura has been replicated in vitro and in vivo through key preclinical work [19, 21, 23,
25, 26, 32]. Furthermore, recent proteomics based approaches continue to produce new
mechanistic insights into the downstream effects of SUMO in contexts such as the re-
sponse to cellular stress, development, and differentiation. Despite that, a clear and com-
prehensive awareness of causal relationships is yet to be defined.
The preclinical literature offers a variety of downstream effects of SUMOylation that
may explain the observed impacts. As with other advances in systems biology, greater
awareness of the panoply of interactions governed by SUMO represents an expanding list
of potentially druggable nodes. A large portion of studies to date have demonstrated the
importance of both SUMO1 [33] and SUMO2/3 [32] in neuroprotection. As SUMO prote-
ases have a central role in neuroprotection, factors regulating the activity of these should
be considered. For example, SENP1 and 3 have been associated with reversible modula-
tion of activity dependent on levels of reactive oxygen species or genotoxic stress, sug-
gesting a possible role as intracellular sentinels for impending insult [20, 34]. The inter-
ested reader is directed to excellent reviews by Droescher et al. and Filippopoulou et al.
covering the variety of both physiologic and protective roles SUMO plays within the cell
[11, 20].

1.2.1 SUMO and the ischemia response

The neuroprotective effects of SUMO regulation are most apparent in the context of

the response to physiologic stressors, including hypoxic and ischemic insults. As with
other aspects of SUMO-related research, the complete breadth of control exerted by
SUMO over the ischemic stress response remains to be comprehensively defined. There
are increasing reports of SUMO-interactions with first- and second-order stress pathways
integral to neuronal survival. As such, SUMO is believed to be a critical intermediary in
optimizing and balancing the various endogenous stress responses to favor regeneration
and repair as opposed to apoptosis and degeneration.
Hypoxia-inducible factor (HIF) is a family of transcription modifier proteins that play
a central role in the hypoxic response, wound healing, and angiogenesis. In the context of
ischemic stroke, HIF activation is associated with increased protection and recovery after
insult [35]. Although HIF expression varies based on ambient oxygen tension and inter-
actions with other pathways such as Nuclear Factor κB (NF-κB), prolonged activation has
been associated with non-scarring tissue regeneration and is a therapeutic target for sev-
eral other pathologies [36]. Unsurprisingly, both SUMOylation and the SUMO pathway
enzymes have been observed to play a role in both positive and negative regulation of the
HIF pathway, with effects varying depending on both the agent and the target of SUMOy-
lation [11]. The differing effects of the family of SUMO-associated E3 ligases is an example
of the former—depending on which ligase is involved, HIF transcription may be posi-
tively regulated, negatively regulated, both, or unaffected. The E3 ligases Cbx4 and PIAS3
positively impact HIF-1α stability and transcription with and without SUMOylation, re-
spectively [37-39]. Alternatively, PIAS1 SUMOylates and inhibits HIF-1B (also known as
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 6 April 2023 doi:10.20944/preprints202304.0094.v1

ARNT) without an appreciable effect on overall HIF transcription [40]. In some cases, op-
posing effects can be produced by the same E3 ligase depending on the target of SUMOy-
lation—PIASγ SUMOylation of HIF-1α negatively impacts HIF stability and transcription,
but PIASγ SUMOylation of pVHL results in increased ubiquitinylation of the same, ulti-
mately resulting in increased HIF stability [41, 42]. Similar modulating SUMO interactions
with the prolyl and asparaginyl hydroxylases that serve as negative feedback to HIF acti-
vation have also been reported: SUMOylation of PHD3 and FIH results in downregulation
(via potentiation of extant negative feedback) and upregulation of HIF transcription [43,
44]. Finally, experiments in HeLa cultures subjected to oxygen-glucose deprivation (OGD)
have demonstrated that changes in SUMOylation in more generalized transcription fac-
tors can inflect the activity of HIF-1—specifically, reduction in SUMO2/3ylation of TFAP2
results in an increase in the transcriptional activity of the same while also enhancing that
of HIF-1 [45].
Whereas activation of HIF-1 is associated with neuroprotection, upregulation of NF-
κB has been observed to correspond with inflammation and neuronal death [46]. Similar
to its impact on HIF, SUMO pathways affect multiple nodes within the NF-κB pathway,
occasionally with contradicting impacts. The known SUMO influences within the path-
way relate primarily to IκBα and the IKK complex [36]. IκBα is a constitutively active
inhibitor that functions by preventing nuclear localization of NF-κB. Experiments with
over-expressed dominant negative murine homologues of Ubc9 observed delays in IκBα
degradation and thus activation of the pathway after in vitro exposure to TNFα, implicat-
ing the SUMO E2 in the endogenous activation of NF-κB [47]. At the same time,
SUMO1ylated IκBα was discovered in COS7 monkey kidney, HEK-293, and Jurkat human
T lymphocyte cultures and was found to be resistant against ubiquitinoylation. Further-
more, the same study found that ubiquitin and SUMO1 competed for the same lysine res-
idue (K21) within IκBα, suggesting competing influences of SUMO and Ubiquitin on IκBα
stability and NF-κB activation [36, 48]. Similarly, SUMO has been found to exert indirect
influence over IκBα by way of the IKK complex. IKK serves to enable nuclear localization
of NF-κB by phosphorylating IκBα. SUMOylation of Annexin-A1 was found to suppress
the NF-κB pathway by increasing IKKα degradation in a microglial model of oxygen-glu-
cose deprivation [49]. On the other hand, SUMO1ylation of IKK-γ (also known as NEMO)
resulted in greater nuclear trafficking and thus activation of NF-κB [50]. The proinflam-
matory impact of SUMO1ylating NEMO was demonstrated in reverse by the observation
that in vitro overexpression of SENP1 resulted in increased de-SUMOylation of NEMO
and concomitant reduction in NF-κB activation [51].
In addition to HIF-1 and NF-κB , SUMO has been found to act as a modulator, albeit
less characterized, of many other cellular pathways. STAT, a signaling pathway involved
in the inflammatory response, was suppressed by SUMOylation of synthetic liver X re-
ceptors (LXR) [52]. SUMOylation of the GluR6 subunit of the Kaianate Receptor (KAR)
has been observed to downregulate the JNK cell-death pathway. Similarly, increased
SUMOylation of the GluR2 subunit of the AMPA receptor has been observed in a murine
model of middle cerebral artery occlusion [53]. Finally, OGD-induced degradation of
SENP3 by PERK has been found to result in increased SUMO2/3ylation of Drp1, resulting
in greater survival during ischemia but increased death during reperfusion [54-56]. As
such, SUMO is integrally linked with the physiological response to ischemia.

 

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