Regulation of mitochondrial division by the Drp1 receptors

Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.

The functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins—Mff, MiD49, and MiD51—play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.

We have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.

To better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.

Studies of dinucleoside monophosphates and monomer-polynucleotide interactions by proton magnetic resonance

The nature of the intra- and intermolecular base-stacking interactions involving several dinucleoside monophosphates in aqueous solution have been investigated by proton magnetic resonance spectrosocopy, and this method has been applied to a study of the interaction of polyuridylic acid with purine and adenosine monomers.

The pmr spectra of adenylyl (3' → 5') cytidine (ApC) and cytidylyl (3' → 5') adenosine (CpA) have been studied as a function of concentration and temperature. The results of these studies indicate that the intramolecular base-stacking interactions between the adenine and cytosine bases of these dinucleoside monophosphates are rather strong, and that the stacking tendencies are comparable for the two sequence isomers. The chemical shifts of the cytosine H₅ and adenine H₂ protons, and their variations with temperature, were shown to be consistent with stacked conformations in which both bases of the dinucleoside monophosphates are preferentially oriented in the anti conformation as in similar dApdC, and dCpdA (dA = deoxyadenosine; dC = deoxycytidine) segments in double helical DNA. The intramolecular stacking interaction was found to have a pronounced effect on the conformations of the ribose moieties, and these conformational changes are discussed. The concentration studies indicate extensive self-association of these dinucleoside monophosphates, and analysis of the concentration data facilitated determination of the dimerization constant for the association process as well as the nature of the intermolecular complexes.

The dependence of the ribose conformation upon the extent of intramolecular base-stacking was used to demonstrate that the base-base interaction in cytidylyl (3' → 5') cytidine (CpC) is rather strong, while there appears to be little interaction between the two uracil bases of uridylyl (3' → 5') uridine (UpU).

Studies of the binding of purine to several ribose and deoxyribose dinucleoside monophosphates show that the mode of interaction is base-stacking, and evidence for the formation of a purine-dinucleoside monophosphate intercalated complex is presented. The purine proton resonances are markedly broadened in this complex, and estimates of the purine linewidths in the complex and the equilibrium constant for purine intercalation are obtained.

A study of the interaction of unsubstitued purine with polyuridylic acid at 29°C by pmr indicated that purine binds to the uracil bases of the polymer by base-stacking. The severe broadening of the purine proton resonances observed provides strong evidence for the intercalation of purine between adjacent uracil bases of poly U. This interaction does not result in a more rigid or ordered structure for the polymer.

Investigation of the interaction between adenosine and polyuridylic acid revealed two modes of interaction between the monomer and the polymer, depending on the temperature. At temperatures above 26°C or so, monomeric adenosine binds to poly U by noncooperative A-U base stacking. Below this temperature, a rigid triple-stranded 1A:2U complex is formed, presumably via cooperative hydrogen-bonding as has previously been reported.

These results clearly illustrate the importance of base-stacking in non-specific interactions between bases, nucleosides and nucleotides, and also reveal the important role of the base-stacking interactions in cooperatively for med structures involving specific base-pairing where both types of interaction are possible.

Investigating the Role of O-GlcNAc Glycosylation in Neurodegeneration

O-GlcNAc glycosylation of nuclear and cytosolic proteins is an essential post-translational modification implicated in many diseases, from cancer to diabetes. Importantly, many important neuronal proteins are also O-GlcNAc modified, and aberrant O-GlcNAcylation of these proteins may contribute to the pathology of neurodegenerative diseases although these mechanisms have not been well defined. Here we investigated the role of O-GlcNAc glycosylation in the brain, utilizing both chemistry and molecular biology to study O-GlcNAc transferase (OGT), the enzyme that adds the sugar modification. To evaluate the role of OGT in adult neurons, we generated a forebrain-specific conditional knockout of OGT (OGT cKO) in mice. Although indistinguishable from wild-type littermates at birth, after three weeks we observe progressive neurodegeneration in OGT cKO mice. Hallmarks of Alzheimer’s disease, including neuronal loss, neuroinflammation, behavioral deficits, hyperphosphorylated tau, and amyloid beta peptide accumulation, are observed. Furthermore, decreases in OGT protein levels were found in human AD brain tissue, suggesting that altered O-GlcNAcylation likely contributes to neurodegenerative diseases in humans. This model is one of a few mouse models that recapitulate AD phenotypes without mutating and overexpressing human tau, amyloid precursor protein, or presenilin, highlighting the essential role of OGT in neurodegenerative pathways.

Given the importance of OGT in the brain, we further investigated the regulation of the OGT enzyme by phosphorylation. We found that phosphorylation of OGT near its C-terminus reduces its activity in cancer cells, and have developed phosphorylation-specific antibodies to aid mechanistic studies. Furthermore, mutation of this phosphorylation site on OGT, followed by overexpression in neurons was shown to enhance neurite outgrowth, demonstrating a functional consequence for this site. Thus phosphorylation of OGT inhibits its activity and enhances neurite outgrowth, and current studies aim to characterize the signaling pathway that regulates OGT phosphorylation in neurons.