MITOCHONDRIAL TRANSPLANTATION AFTER SPINAL CORD INJURY: EFFECTS ON TISSUE BIOENERGETICS AND FUNCTIONAL NEUROPROTECTION

Contusion spinal cord injury (SCI) results in devastating life-long debilitation in which there are currently no effective treatments. The primary injury site presents a complex environment marked by subsequent secondary pathophysiological cascades involving excessive reactive oxygen and nitrogen species (ROS/RNS) production, glutamate-induced excitotoxicity, calcium dysregulation, and delayed neuronal apoptosis. Many of these cascades involve mitochondrial dysfunction, thus a single mitochondrial-centric therapy that targets a variety of these factors could be far reaching in its potential benefits after SCI. As such, this dissertation examines whether transplantation of exogenous mitochondria after SCI can attenuate secondary injury cascades to decrease the spread and severity of the injury. Our first experiment tested the dose-dependent effects of mitochondrial transplantation on the ability to maintain acute overall bioenergetics after SCI. We compared transplantation of mitochondria originating from two different sources-cultured PC12 cells or rat soleus leg muscle. 24 hours after injury, State III oxygen consumption rates were maintained to over 80% of sham levels when 100ug of mitochondria was transplanted, regardless of the origin of the mitochondria. Complex I enzyme activity assays corroborated our findings that the 100ug dosage gave optimal benefits compared to vehicle injection. We also analyzed the rostral-caudal distribution and cell-type colocalization of transplanted transgenically-labeled tGFP mitochondria after SCI. There were greater volumes and rostral-caudal spread of tGFP mitochondria at the 24 hour time point compared to 7 days post injection. tGFP mitochondria had the greatest propensity to colocalize with macrophages and pericytes. Colocalization was evident in endothelial cells, oligodendrocytes and astrocytes, though no such colabeling was present in neurons. Further, colocalization of tGFP was always greater at the 24 hour time compared to 48 hour or 7days post injection time points. These data indicate that there is a cell-type difference in incorporation potential of exogenous mitochondria which changes over time. Finally, we tested the effects of mitochondrial transplantation on long term functional recovery. Animals were injected with either vehicle, 100ug cell-derived mitochondria, or 100ug muscle-derived mitochondria immediately after contusion SCI. Functional analyses including BBB overground locomotor scale and von Frey mechanical sensitivity tests did not show any differences between treatment groups. Likewise, there were no differences in tissue sparing when mitochondria were transplanted compared to vehicle injections, though there were higher neuronal cell counts in tGFP mitochondria injected groups caudal of the injury site. These studies present the potential of mitochondrial transplantation for therapeutic intervention after SCI. While our acute measures do not correspond into long term recovery, we show that at 24 hours transplanted mitochondria do have an effect on bioenergetics and that they are taken into host cells. We believe that further investigation into caveats and technical refinement is necessary at this time to translate the evident acute bioenergetic recovery into long term functional recovery

Mitochondrial Transplantation Strategies as Potential Therapeutics for Central Nervous System Trauma

Mitochondria are essential cellular organelles critical for generating adenosine triphosphate for cellular homeostasis, as well as various mechanisms that can lead to both necrosis and apoptosis. The field of “mitochondrial medicine” is emerging in which injury/disease states are targeted therapeutically at the level of the mitochondrion, including specific antioxidants, bioenergetic substrate additions, and membrane uncoupling agents. Consequently, novel mitochondrial transplantation strategies represent a potentially multifactorial therapy leading to increased adenosine triphosphate production, decreased oxidative stress, mitochondrial DNA replacement, improved bioenergetics and tissue sparing. Herein, we describe briefly the history of mitochondrial transplantation and the various techniques used for both in vitro and in vivo delivery, the benefits associated with successful transference into both peripheral and central nervous system tissues, along with caveats and pitfalls that hinder the advancements of this novel therapeutic

Optimization of Mitochondrial Isolation Techniques for Intraspinal Transplantation Procedures

Background—Proper mitochondrial function is essential to maintain normal cellular bioenergetics and ionic homeostasis. In instances of severe tissue damage, such as traumatic brain and spinal cord injury, mitochondria become damaged and unregulated leading to cell death. The relatively unexplored field of mitochondrial transplantation following neurotrauma is based on the theory that replacing damaged mitochondria with exogenous respiratory-competent mitochondria can restore overall tissue bioenergetics. New Method—We optimized techniques in vitro to prepare suspensions of isolated mitochondria for transplantation in vivo. Mitochondria isolated from cell culture were genetically labeled with turbo-green fluorescent protein (tGFP) for imaging and tracking purposes in vitro and in vivo. Results—We used time-lapse confocal imaging to reveal the incorporation of exogenous fluorescently-tagged mitochondria into PC-12 cells after brief co-incubation. Further, we show that mitochondria can be injected into the spinal cord with immunohistochemical evidence of host cellular uptake within 24 hours. Comparison to Existing Methods—Our methods utilize transgenic fluorescent labeling of mitochondria for a nontoxic and photostable alternative to other labeling methods. Substrate addition to isolated mitochondria helped to restore state III respiration at room temperature prior to transplantation. These experiments delineate refined methods to use transgenic cell lines for the purpose of isolating well coupled mitochondria that have a permanent fluorescent label that allows real time tracking of transplanted mitochondria in vitro, as well as imaging in situ. Conclusions—These techniques lay the foundation for testing the potential therapeutic effects of mitochondrial transplantation following spinal cord injury and other animal models of neurotrauma

Pioglitazone Treatment Following Spinal Cord Injury Maintains Acute Mitochondrial Integrity and Increases Chronic Tissue Sparing and Functional Recovery

Pioglitazone is an FDA-approved PPAR-γ agonist drug used to for treat diabetes, and it has demonstrated neuroprotective effects in multiple models of central nervous system (CNS) injury. Acute treatment after spinal cord injury (SCI) in rats is reported to suppress neuroinflammation, rescue injured tissues, and improve locomotor recovery. In the current study, we additionally assessed the protective efficacy of pioglitazone treatment on acute mitochondrial respiration, as well as functional and anatomical recovery after contusion SCI in adult male C57BL/6 mice. Mice received either vehicle or pioglitazone (10 mg/kg) at either 15 min or 3 hr after injury (75 kDyn at T9) followed by a booster at 24 hr post-injury. At 25 hr, mitochondria were isolated from spinal cord segments centered on the injury epicenters and assessed for their respiratory capacity. Results showed significantly compromised mitochondrial respiration 25 hr following SCI, but pioglitazone treatment that was initiated either at 15 min or 3 hr post-injury significantly maintained mitochondrial respiration rates near sham levels. A second cohort of injured mice received pioglitazone at 15 min post injury, then once a day for 5 days post-injury to assess locomotor recovery and tissue sparing over 4 weeks. Compared to vehicle, pioglitazone treatment resulted in significantly greater recovery of hind-limb function over time, as determined by serial locomotor BMS assessments and both terminal BMS subscores and gridwalk performance. Such improvements correlated with significantly increased grey and white matter tissue sparing, although pioglitazone treatment did not abrogate long-term injury-induced inflammatory microglia/macrophage responses. In sum, pioglitazone significantly increased functional neuroprotection that was associated with remarkable maintenance of acute mitochondrial bioenergetics after traumatic SCI. This sets the stage for dose-response and delayed administration studies to maximize pioglitazone’s efficacy for SCI while elucidating the precise role that mitochondria play in governing its neuroprotection; the ultimate goal to develop novel therapeutics that specifically target mitochondrial dysfunction

Q134R: Small chemical compound with NFAT inhibitory properties improves behavioral performance and synapse function in mouse models of amyloid pathology

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer's disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (= 3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal A beta plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders

Q134R: Small Chemical Compound with NFAT Inhibitory Properties Improves Behavioral Performance and Synapse Function in Mouse Models of Amyloid Pathology

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer\u27s disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild-type mice (3–4 months old) infused with oligomeric Aβ peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders

sj-pdf-1-jcb-10.1177_0271678X231216142 - Supplemental material for Using digital pathology to analyze the murine cerebrovasculature

Supplemental material, sj-pdf-1-jcb-10.1177_0271678X231216142 for Using digital pathology to analyze the murine cerebrovasculature by Dana M Niedowicz, Jenna L Gollihue, Erica M Weekman, Panhavuth Phe, Donna M Wilcock, Christopher M Norris and Peter T Nelson in Journal of Cerebral Blood Flow & Metabolism</p