Deletion of a Neuronal Drp1 Activator Protects against Cerebral Ischemia.

Mitochondrial fission catalyzed by dynamin-related protein 1 (Drp1) is necessary for mitochondrial biogenesis and maintenance of healthy mitochondria. However, excessive fission has been associated with multiple neurodegenerative disorders, and we recently reported that mice with smaller mitochondria are sensitized to ischemic stroke injury. Although pharmacological Drp1 inhibition has been put forward as neuroprotective, the specificity and mechanism of the inhibitor used is controversial. Here, we provide genetic evidence that Drp1 inhibition is neuroprotective. Drp1 is activated by dephosphorylation of an inhibitory phosphorylation site, Ser637. We identify Bß2, a mitochondria-localized protein phosphatase 2A (PP2A) regulatory subunit, as a neuron-specific Drp1 activator in vivo Bß2 KO mice of both sexes display elongated mitochondria in neurons and are protected from cerebral ischemic injury. Functionally, deletion of Bß2 and maintained Drp1 Ser637 phosphorylation improved mitochondrial respiratory capacity, Ca2+ homeostasis, and attenuated superoxide production in response to ischemia and excitotoxicity in vitro and ex vivo Last, deletion of Bß2 rescued excessive stroke damage associated with dephosphorylation of Drp1 S637 and mitochondrial fission. These results indicate that the state of mitochondrial connectivity and PP2A/Bß2-mediated dephosphorylation of Drp1 play a critical role in determining the severity of cerebral ischemic injury. Therefore, Bß2 may represent a target for prophylactic neuroprotective therapy in populations at high risk of stroke.SIGNIFICANCE STATEMENT With recent advances in clinical practice including mechanical thrombectomy up to 24 h after the ischemic event, there is resurgent interest in neuroprotective stroke therapies. In this study, we demonstrate reduced stroke damage in the brain of mice lacking the Bß2 regulatory subunit of protein phosphatase 2A, which we have shown previously acts as a positive regulator of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1). Importantly, we provide evidence that deletion of Bß2 can rescue excessive ischemic damage in mice lacking the mitochondrial PKA scaffold AKAP1, apparently via opposing effects on Drp1 S637 phosphorylation. These results highlight reversible phosphorylation in bidirectional regulation of Drp1 activity and identify Bß2 as a potential pharmacological target to protect the brain from stroke injury.

Mutant huntingtin impairs PNKP and ATXN3, disrupting DNA repair and transcription.

How huntingtin (HTT) triggers neurotoxicity in Huntington's disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.

AKAP1 Protects from Cerebral Ischemic Stroke by Inhibiting Drp1-Dependent Mitochondrial Fission.

Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1-/- mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex II of the electron transport chain, increases superoxide production, and impairs Ca2+ homeostasis in neurons subjected to excitotoxic glutamate. Ca2+ deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drp1-dependent mitochondrial fission by the outer mitochondrial AKAP1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca2+ deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration.SIGNIFICANCE STATEMENT Previous work suggests that activation of dynamin-related protein 1 (Drp1) and mitochondrial fission contribute to ischemic injury in the brain. However, the specificity and efficacy of the pharmacological Drp1 inhibitor mdivi-1 that was used has now been discredited by several high-profile studies. Our report is timely and highly impactful because it provides the first evidence that genetic disinhibition of Drp1 via knock-out of the mitochondrial protein kinase A (PKA) scaffold AKAP1 exacerbates stroke injury in mice. Mechanistically, we show that electron transport deficiency, increased superoxide production, and Ca2+ overload result from genetic disinhibition of Drp1. In summary, our work settles current controversies regarding the role of mitochondrial fission in neuronal injury, provides mechanisms, and suggests that fission inhibitors hold promise as future therapeutic agents.

Fasting Activates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Function during Homeostasis and Aging.

Diet has a profound effect on tissue regeneration in diverse organisms, and low caloric states such as intermittent fasting have beneficial effects on organismal health and age-associated loss of tissue function. The role of adult stem and progenitor cells in responding to short-term fasting and whether such responses improve regeneration are not well studied. Here we show that a 24 hr fast augments intestinal stem cell (ISC) function in young and aged mice by inducing a fatty acid oxidation (FAO) program and that pharmacological activation of this program mimics many effects of fasting. Acute genetic disruption of Cpt1a, the rate-limiting enzyme in FAO, abrogates ISC-enhancing effects of fasting, but long-term Cpt1a deletion decreases ISC numbers and function, implicating a role for FAO in ISC maintenance. These findings highlight a role for FAO in mediating pro-regenerative effects of fasting in intestinal biology, and they may represent a viable strategy for enhancing intestinal regeneration.

Therapy development in Huntington disease: From current strategies to emerging opportunities.

Huntington disease (HD) is a progressive autosomal dominant neurodegenerative disorder in which patients typically present with uncontrolled involuntary movements and subsequent cognitive decline. In 1993, a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene was identified as the cause of this disorder. This extended CAG repeat results in production of HTT protein with an expanded polyglutamine tract, leading to pathogenic HTT protein conformers that are resistant to protein turnover, culminating in cellular toxicity and neurodegeneration. Research into the mechanistic basis of HD has highlighted a role for bioenergetics abnormalities stemming from mitochondrial dysfunction, and for synaptic defects, including impaired neurotransmission and excitotoxicity. Interference with transcription regulation may underlie the mitochondrial dysfunction. Current therapies for HD are directed at treating symptoms, as there are no disease-modifying therapies. Commonly prescribed drugs for involuntary movement control include tetrabenazine, a potent and selective inhibitor of vesicular monoamine transporter 2 that depletes synaptic monoamines, and olanzapine, an atypical neuroleptic that blocks the dopamine D2 receptor. Various drugs are used to treat non-motor features. The HD therapeutic pipeline is robust, as numerous efforts are underway to identify disease-modifying treatments, with some small compounds and biological agents moving into clinical trials. Especially encouraging are dosage reduction strategies, including antisense oligonucleotides, and molecules directed at transcription dysregulation. Given the depth and breadth of current HD drug development efforts, there is reason to believe that disease-modifying therapies for HD will emerge, and this achievement will have profound implications for the entire neurotherapeutics field.

PPARδ activation by bexarotene promotes neuroprotection by restoring bioenergetic and quality control homeostasis.

Neurons must maintain protein and mitochondrial quality control for optimal function, an energetically expensive process. The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that promote mitochondrial biogenesis and oxidative metabolism. We recently determined that transcriptional dysregulation of PPARδ contributes to Huntington's disease (HD), a progressive neurodegenerative disorder resulting from a CAG-polyglutamine repeat expansion in the huntingtin gene. We documented that the PPARδ agonist KD3010 is an effective therapy for HD in a mouse model. PPARδ forms a heterodimer with the retinoid X receptor (RXR), and RXR agonists are capable of promoting PPARδ activation. One compound with potent RXR agonist activity is the U.S. Food and Drug Administration-approved drug bexarotene. We tested the therapeutic potential of bexarotene in HD and found that bexarotene was neuroprotective in cellular models of HD, including medium spiny-like neurons generated from induced pluripotent stem cells (iPSCs) derived from patients with HD. To evaluate bexarotene as a treatment for HD, we treated the N171-82Q mouse model with the drug and found that bexarotene improved motor function, reduced neurodegeneration, and increased survival. To determine the basis for PPARδ neuroprotection, we evaluated metabolic function and noted markedly impaired oxidative metabolism in HD neurons, which was rescued by bexarotene or KD3010. We examined mitochondrial and protein quality control in cellular models of HD and observed that treatment with a PPARδ agonist promoted cellular quality control. By boosting cellular activities that are dysfunctional in HD, PPARδ activation may have therapeutic applications in HD and potentially other neurodegenerative diseases.

PPAR-δ is repressed in Huntington's disease, is required for normal neuronal function and can be targeted therapeutically.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which encodes a polyglutamine tract in the HTT protein. We found that peroxisome proliferator-activated receptor delta (PPAR-δ) interacts with HTT and that mutant HTT represses PPAR-δ-mediated transactivation. Increased PPAR-δ transactivation ameliorated mitochondrial dysfunction and improved cell survival of neurons from mouse models of HD. Expression of dominant-negative PPAR-δ in the central nervous system of mice was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities and transcriptional alterations that recapitulated HD-like phenotypes. Expression of dominant-negative PPAR-δ specifically in the striatum of medium spiny neurons in mice yielded HD-like motor phenotypes, accompanied by striatal neuron loss. In mouse models of HD, pharmacologic activation of PPAR-δ using the agonist KD3010 improved motor function, reduced neurodegeneration and increased survival. PPAR-δ activation also reduced HTT-induced neurotoxicity in vitro and in medium spiny-like neurons generated from stem cells derived from individuals with HD, indicating that PPAR-δ activation may be beneficial in HD and related disorders.

PKA/AKAP1 and PP2A/Bß2 regulate neuronal morphogenesis via Drp1 phosphorylation and mitochondrial bioenergetics.

Mitochondrial shape is determined by fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by dynamin-related protein 1 (Drp1), a large GTPase of the dynamin family that is highly expressed in neurons and regulated by various posttranslational modifications, including phosphorylation. We report here that reversible phosphorylation of Drp1 at a conserved Ser residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase (PP2A/Bß2) impacts dendrite and synapse development in cultured rat hippocampal neurons. PKA/AKAP1-mediated phosphorylation of Drp1 at Ser656 increased mitochondrial length and dendrite occupancy, enhancing dendritic outgrowth but paradoxically decreasing synapse number and density. Opposing PKA/AKAP1, PP2A/Bß2-mediated dephosphorylation of Drp1 at Ser656 fragmented and depolarized mitochondria and depleted them from dendrites, stunting dendritic outgrowth but augmenting synapse formation. Raising and lowering intracellular calcium reproduced the effects of dephospho-Drp1 and phospho-Drp1on dendrite and synapse development, respectively, while boosting mitochondrial membrane potential with l-carnitine-fostered dendrite at the expense of synapse formation without altering mitochondrial size or distribution. Thus, outer mitochondrial PKA and PP2A regulate neuronal development by inhibiting and promoting mitochondrial division, respectively. We propose that the bioenergetic state of mitochondria, rather than their localization or shape per se, is the key effector of Drp1, altering calcium homeostasis to modulate neuronal morphology and connectivity.

Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1.

Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults.