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Cambridge, MA, United States

Cesani M.,San Raffaele Scientific Institute | Cesani M.,Harvard University | Cavalca E.,San Raffaele Scientific Institute | Macco R.,Vita-Salute San Raffaele University | And 16 more authors.
Annals of Neurology | Year: 2014

Objective To facilitate development of novel disease-modifying therapies for lysosomal storage disorder (LSDs) characterized by nervous system involvement such as metachromatic leukodystrophy (MLD), molecular markers for monitoring disease progression and therapeutic response are needed. To this end, we sought to identify blood transcripts associated with the progression of MLD. Methods Genome-wide expression analysis was performed in primary T lymphocytes of 24 patients with MLD compared to 24 age- and sex-matched healthy controls. Genes associated with MLD were identified, confirmed on a quantitative polymerase chain reaction platform, and replicated in an independent patient cohort. mRNA and protein expression of the prioritized gene family of metallothioneins was evaluated in postmortem patient brains and in mouse models representing 6 other LSDs. Metallothionein expression during disease progression and in response to specific treatment was evaluated in 1 of the tested LSD mouse models. Finally, a set of in vitro studies was planned to dissect the biological functions exerted by this class of molecules. Results Metallothionein genes were significantly overexpressed in T lymphocytes and brain of patients with MLD and generally marked nervous tissue damage in the LSDs here evaluated. Overexpression of metallothioneins correlated with measures of disease progression in mice and patients, whereas their levels decreased in mice upon therapeutic treatment. In vitro studies indicated that metallothionein expression is regulated in response to oxidative stress and inflammation, which are biochemical hallmarks of lysosomal storage diseases. Interpretation Metallothioneins are potential markers of neurologic disease processes and treatment response in LSDs.Ann Neurol 2014;75:127-137 © 2013 Child Neurology Society/American Neurological Association. Source

Liu M.,Harvard NeuroDiscovery Center | Girma E.,Harvard NeuroDiscovery Center | Glicksman M.A.,Harvard NeuroDiscovery Center | Stein R.L.,Sirtris Pharmaceuticals
Biochemistry | Year: 2010

Cdk5/p25 is a member of the cyclin-dependent, Ser/Thr kinase family and has been identified as one of the principle Alzheimers disease-associated kinases that promote the formation of hyperphosphorylated tau, the major component of neurofibrillary tangles. We and others have been developing inhibitors of cdk5/p25 as possible therapeutic agents for Alzheimers disease (AD). In support of these efforts, we examine the metal effect and solvent kinetic isotope effect on cdk5/p25-catalyzed H1P (a histone H-1-derived peptide) phosphorylation. Here, we report that a second Mg2+ in addition to the one forming the MgATP complex is required to bind to cdk5/p25 for its catalytic activity. It activates cdk5/p25 by demonstrating an increase in kcat and induces a conformational change that favors ATP binding but has no effect on the binding affinity for the H1P peptide substrate. The binding of the second Mg 2+ does not change the binding order of substrates. The reaction follows the same rapid equilibrium random mechanism in the presence or absence of the second Mg2+ as evidenced by initial velocity analysis and substrate analogue and product inhibition studies. A linear proton inventory with a normal SKIE of 2.0 ± 0.1 in the presence of the second Mg 2+ was revealed and suggested a single proton transfer in the rate-limiting phosphoryl transfer step. The pH profile revealed a residue with a pKa of 6.5 that is most likely the general acid-base catalyst facilitating the proton transfer. © 2010 American Chemical Society. Source

Liu M.,Harvard NeuroDiscovery Center | Dobson B.,Harvard NeuroDiscovery Center | Glicksman M.A.,Harvard NeuroDiscovery Center | Yue Z.,Mount Sinai School of Medicine | Stein R.L.,Sirtris Pharmaceuticals
Biochemistry | Year: 2010

Recent studies have identified mutations in the leucine-rich repeat kinase2 gene (LRRK2) in the most common familial forms and some sporadic forms of Parkinson's disease (PD). LRRK2 is a large and complex protein that possesses kinase and GTPase activities. Some LRRK2 mutants enhance kinase activity and possibly contribute to PD through a toxic gain-of-function mechanism. Given the role of LRRK2 in the pathogenesis of PD, understanding the kinetic mechanism of its two enzymatic properties is critical for the discovery of inhibitors of LRRK2 kinase that would be therapeutically useful in treating PD. In this report, by using LRRK2 protein purified from murine brain, first we characterize kinetic mechanisms for the LRRK2-catalyzed phosphorylation of two peptide substrates: PLK-derived peptide (PLK-peptide) and LRRKtide. We found that LRRK2 follows a rapid equilibrium random mechanism for the phosphorylation of PLK-peptide with either ATP or PLK-peptide being the first substrate binding to the enzyme, as evidenced by initial velocity and inhibition mechanism studies with nucleotide analogues AMP and AMP-PNP, product ADP, and an analogue of the peptide substrate. The binding of the first substrate has no effect on the binding affinity of the second substrate. Identical mechanistic conclusions were drawn when LRRKtide was the phosphoryl acceptor. Next, we characterize the GTPase activity of LRRK2 with a kcat of 0.2 ± 0.02 s -1 and a Km of 210 ± 29 μM. A SKIE of 0.97 ± 0.04 was measured on kcat for the GTPase activity of LRRK2 in a D2O molar fraction of 0.86 and suggested that the product dissociation step is rate-limiting, of the steps governed by kcat in the LRRK2-catalyzed GTP hydrolysis. Surprisingly, binding of GTP, GDP, or GMP has no effect on kinase activity, although GMP and GDP inhibit the GTPase activity. Finally, we have identified compound LDN-73794 through screen of LRRK2 kinase inhibitors. Our study revealed that this compound is a competitive inhibitor of the binding of ATP and inhibits the kinase activity without affecting the GTPase activity. © 2010 American Chemical Society. Source

Androutsellis-Theotokis A.,U.S. National Institutes of Health | Androutsellis-Theotokis A.,Academy of Athens | Rueger M.A.,U.S. National Institutes of Health | Rueger M.A.,University of Cologne | And 12 more authors.
PLoS ONE | Year: 2010

Background: The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools [1,2,3,4,5,6]. Methodology/Principal Findings: Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes. Conclusions/Significance: We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration. Source

Ray S.,Harvard NeuroDiscovery Center | Bender S.,Harvard NeuroDiscovery Center | Bender S.,Harvard University | Kang S.,Harvard NeuroDiscovery Center | And 7 more authors.
Journal of Biological Chemistry | Year: 2014

The effect of leucine-rich repeat kinase 2 (LRRK2) mutation I2020T on its kinase activity has been controversial, with both increased and decreased effects being reported. We conducted steady-state and pre-steady-state kinetic studies on LRRKtide and its analog LRRKtideS. Their phosphorylation differs by the rate-limiting steps: product release is rate-limiting for LRRKtide and phosphoryl transfer is rate-limiting for LRRKtideS. As a result, we observed that the I2020T mutant is more active than wild type (WT) LRRK2 for LRRKtideS phosphorylation, whereas it is less active than WT for LRRKtide phosphorylation. Our pre-steady-state kinetic data suggest that (i) the I2020T mutant accelerates the rates of phosphoryl transfer of both reactions by 3-7-fold; (ii) this increase is masked by a rate-limiting product release step for LRRKtide phosphorylation; and (iii) the observed lower activity of the mutant for LRRKtide phosphorylation is a consequence of its instability: the concentration of the active form of the mutant is 3-fold lower than WT. The I2020T mutant has a dramatically low KATP and therefore leads to resistance toATPcompetitive inhibitors.Twowell known DFGout or type II inhibitors are also weaker toward the mutant because they inhibit the mutant in an unexpected ATP competitive mechanism. The I2020 residue lies next to the DYGmotif of the activation loop of the LRRK2 kinase domain. Our modeling and metadynamic simulations suggest that the I2020T mutant stabilizes the DYG-in active conformation and creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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