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mTOR Inhibition with Sirolimus in Multiple System Atrophy: A Randomized, Double-Blind, Placebo-Controlled Futility Trial and 1-Year Biomarker Longitudinal Analysis

Palma, Jose-Alberto; Martinez, Jose; Millar Vernetti, Patricio; Ma, Thong; Perez, Miguel A; Zhong, Judy; Qian, Yingzhi; Dutta, Suman; Maina, Katherine N; Siddique, Ibrar; Bitan, Gal; Ades-Aron, Benjamin; Shepherd, Timothy M; Kang, Un J; Kaufmann, Horacio
BACKGROUND:Multiple system atrophy (MSA) is a fatal neurodegenerative disease characterized by the aggregation of α-synuclein in glia and neurons. Sirolimus (rapamycin) is an mTOR inhibitor that promotes α-synuclein autophagy and reduces its associated neurotoxicity in preclinical models. OBJECTIVE:To investigate the efficacy and safety of sirolimus in patients with MSA using a futility design. We also analyzed 1-year biomarker trajectories in the trial participants. METHODS:Randomized, double-blind, parallel group, placebo-controlled clinical trial at the New York University of patients with probable MSA randomly assigned (3:1) to sirolimus (2-6 mg daily) for 48 weeks or placebo. Primary endpoint was change in the Unified MSA Rating Scale (UMSARS) total score from baseline to 48 weeks. ( NCT03589976). RESULTS:The trial was stopped after a pre-planned interim analysis met futility criteria. Between August 15, 2018 and November 15, 2020, 54 participants were screened, and 47 enrolled and randomly assigned (35 sirolimus, 12 placebo). Of those randomized, 34 were included in the intention-to-treat analysis. There was no difference in change from baseline to week 48 between the sirolimus and placebo in UMSARS total score (mean difference, 2.66; 95% CI, -7.35-6.91; P = 0.648). There was no difference in UMSARS-1 and UMSARS-2 scores either. UMSARS scores changes were similar to those reported in natural history studies. Neuroimaging and blood biomarker results were similar in the sirolimus and placebo groups. Adverse events were more frequent with sirolimus. Analysis of 1-year biomarker trajectories in all participants showed that increases in blood neurofilament light chain (NfL) and reductions in whole brain volume correlated best with UMSARS progression. CONCLUSIONS:Sirolimus for 48 weeks was futile to slow the progression of MSA and had no effect on biomarkers compared to placebo. One-year change in blood NfL and whole brain atrophy are promising biomarkers of disease progression for future clinical trials. © 2022 International Parkinson and Movement Disorder Society.
PMID: 35040506
ISSN: 1531-8257
CID: 5131432

A rapid α-synuclein seed assay of Parkinson's disease CSF panel shows high diagnostic accuracy

Orrù, Christina D; Ma, Thong C; Hughson, Andrew G; Groveman, Bradley R; Srivastava, Ankit; Galasko, Douglas; Angers, Rachel; Downey, Patrick; Crawford, Karen; Hutten, Samantha J; Kang, Un Jung; Caughey, Byron
BACKGROUND:Assays that specifically measure α-synuclein seeding activity in biological fluids could revolutionize the diagnosis of Parkinson's disease. Recent improvements in α-synuclein real-time quaking-induced conversion assays of cerebrospinal fluid have dramatically reduced reaction times from 5-13 days down to 1-2 days. OBJECTIVE:To test our improved assay against a panel of cerebrospinal fluid specimens from patients with Parkinson's disease and healthy controls from the MJ Fox Foundation/NINDS BioFIND collection. METHODS:Specimens collected from healthy controls and patients with clinically typical moderate-to-advanced Parkinson's disease were tested without prior knowledge of disease status. Correlative analyses between assay parameters and clinical measures were performed by an independent investigator. RESULTS:BioFIND samples gave positive signals in 105/108 (97%) Parkinson's disease cases versus 11/85 (13%) healthy controls. Receiver operating characteristic analyses of diagnosis of cases versus healthy controls gave areas under the curve of 95%. Beyond binary positive/negative determinations, only weak correlations were observed between various assay response parameters and Parkinson's disease clinical measures or other cerebrospinal fluid analytes. Of note, REM sleep behavioral disorder questionnaire scores correlated with the reaction times needed to reach 50% maximum fluorescence. Maximum fluorescence was inversely correlated with Unified Parkinson's Disease Rating Scale motor scores, which was driven by the patients without REM sleep behavioral disorder. CONCLUSIONS:Our improved α-synuclein seed amplification assay dramatically reduces the time needed to diagnose Parkinson's disease while maintaining the high-performance standards associated with previous α-synuclein seed assays, supporting the clinical utility of this assay for Parkinson's disease diagnosis.
PMID: 33373501
ISSN: 2328-9503
CID: 4765002

Alterations in the intrinsic properties of striatal cholinergic interneurons after dopamine lesion and chronic L-DOPA

Choi, Se Joon; Ma, Thong C; Ding, Yunmin; Cheung, Timothy; Joshi, Neal; Sulzer, David; Mosharov, Eugene V; Kang, Un Jung
Changes in striatal cholinergic interneuron (ChI) activity are thought to contribute to Parkinson's disease pathophysiology and dyskinesia from chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment, but the physiological basis of these changes is unknown. We find that dopamine lesion decreases the spontaneous firing rate of ChIs, whereas chronic treatment with L-DOPA of lesioned mice increases baseline ChI firing rates to levels beyond normal activity. The effect of dopamine loss on ChIs was due to decreased currents of both hyperpolarization-activated cyclic nucleotide-gated (HCN) and small conductance calcium-activated potassium (SK) channels. L-DOPA reinstatement of dopamine normalized HCN activity, but SK current remained depressed. Pharmacological blockade of HCN and SK activities mimicked changes in firing, confirming that these channels are responsible for the molecular adaptation of ChIs to dopamine loss and chronic L-DOPA treatment. These findings suggest that targeting ChIs with channel-specific modulators may provide therapeutic approaches for alleviating L-DOPA-induced dyskinesia in PD patients.
PMID: 32687053
ISSN: 2050-084x
CID: 4533332

Comparative study of cerebrospinal fluid α-synuclein seeding aggregation assays for diagnosis of Parkinson's disease

Kang, Un Jung; Boehme, Amelia K; Fairfoul, Graham; Shahnawaz, Mohammad; Ma, Thong Chi; Hutten, Samantha J; Green, Alison; Soto, Claudio
BACKGROUND:PD diagnosis is based primarily on clinical criteria and can be inaccurate. Biological markers, such as α-synuclein aggregation, that reflect ongoing pathogenic processes may increase diagnosis accuracy and allow disease progression monitoring. Though α-synuclein aggregation assays have been published, reproducibility, standardization, and validation are key challenges for their development as clinical biomarkers. OBJECTIVE:To cross-validate two α-synuclein seeding aggregation assays developed to detect pathogenic oligomeric α-synuclein species in CSF using samples from the same PD patients and healthy controls from the BioFIND cohort. METHODS:CSF samples were tested by two independent laboratories in a blinded fashion. BioFIND features standardized biospecimen collection of clinically typical moderate PD patients and nondisease controls. α-synuclein aggregation was measured by protein misfolding cyclic amplification (Soto lab) and real-time quaking-induced conversion (Green lab). Results were analyzed by an independent statistician. RESULTS:Measuring 105 PD and 79 healthy control CSF samples, these assays showed 92% concordance. The areas under the curve from receiver operating characteristic curve analysis for the diagnosis of PD versus healthy controls were 0.93 for protein misfolding cyclic amplification, 0.89 for real-time quaking-induced conversion, and 0.95 when considering only concordant assay results. Clinical characteristics of false-positive and -negative subjects were not different from true-negative and -positive subjects, respectively. CONCLUSIONS:These α-synuclein seeding aggregation assays are reliable and reproducible for PD diagnosis. Assay parameters did not correlate with clinical parameters, including disease severity or duration. This assay is highly accurate for PD diagnosis and may impact clinical practice and clinical trials. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.
PMID: 30840785
ISSN: 1531-8257
CID: 3733802

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations

Li, Hongyu; Ham, Ahrom; Ma, Thong Chi; Kuo, Sheng-Han; Kanter, Ellen; Kim, Donghoon; Ko, Han Seok; Quan, Yi; Sardi, Sergio Pablo; Li, Aiqun; Arancio, Ottavio; Kang, Un Jung; Sulzer, David; Tang, Guomei
Heterozygous mutations in GBA, the gene encoding the lysosomal enzyme glucosylceramidase beta/β-glucocerebrosidase, comprise the most common genetic risk factor for Parkinson disease (PD), but the mechanisms underlying this association remain unclear. Here, we show that in GbaL444P/WT knockin mice, the L444P heterozygous Gba mutation triggers mitochondrial dysfunction by inhibiting autophagy and mitochondrial priming, two steps critical for the selective removal of dysfunctional mitochondria by autophagy, a process known as mitophagy. In SHSY-5Y neuroblastoma cells, the overexpression of L444P GBA impeded mitochondrial priming and autophagy induction when endogenous lysosomal GBA activity remained intact. By contrast, genetic depletion of GBA inhibited lysosomal clearance of autophagic cargo. The link between heterozygous GBA mutations and impaired mitophagy was corroborated in postmortem brain tissue from PD patients carrying heterozygous GBA mutations, where we found increased mitochondrial content, mitochondria oxidative stress and impaired autophagy. Our findings thus suggest a mechanistic basis for mitochondrial dysfunction associated with GBA heterozygous mutations. Abbreviations: AMBRA1: autophagy/beclin 1 regulator 1; BECN1: beclin 1, autophagy related; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chloroyphenylhydrazone; CYCS: cytochrome c, somatic; DNM1L/DRP1: dynamin 1-like; ER: endoplasmic reticulum; GBA: glucosylceramidase beta; GBA-PD: Parkinson disease with heterozygous GBA mutations; GD: Gaucher disease; GFP: green fluorescent protein; LC3B: microtubule-associated protein 1 light chain 3 beta; LC3B-II: lipidated form of microtubule-associated protein 1 light chain 3 beta; MitoGreen: MitoTracker Green; MitoRed: MitoTracker Red; MMP: mitochondrial membrane potential; MTOR: mechanistic target of rapamycin kinase; MYC: MYC proto-oncogene, bHLH transcription factor; NBR1: NBR1, autophagy cargo receptor; Non-GBA-PD: Parkinson disease without GBA mutations; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RFP: red fluorescent protein; ROS: reactive oxygen species; SNCA: synuclein alpha; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1/Porin: voltage dependent anion channel 1; WT: wild type.
PMID: 30160596
ISSN: 1554-8635
CID: 3501942

Dopamine neuron glutamate cotransmission evokes a delayed excitation in lateral dorsal striatal cholinergic interneurons

Chuhma, Nao; Mingote, Susana; Yetnikoff, Leora; Kalmbach, Abigail; Ma, Thong; Ztaou, Samira; Sienna, Anna-Claire; Tepler, Sophia; Poulin, Jean-Francois; Ansorge, Mark; Awatramani, Rajeshwar; Kang, Un Jung; Rayport, Stephen
Dopamine neurons have different synaptic actions in the ventral and dorsal striatum (dStr), but whether this heterogeneity extends to dStr subregions has not been addressed. We have found that optogenetic activation of dStr dopamine neuron terminals in mouse brain slices pauses the firing of cholinergic interneurons in both the medial and lateral subregions, while in the lateral subregion the pause is shorter due to a subsequent excitation. This excitation is mediated mainly by metabotropic glutamate receptor 1 (mGluR1) and partially by dopamine D1-like receptors coupled to transient receptor potential channel 3 and 7. DA neurons do not signal to spiny projection neurons in the medial dStr, while they elicit ionotropic glutamate responses in the lateral dStr. The DA neurons mediating these excitatory signals are in the substantia nigra (SN). Thus, SN dopamine neurons engage different receptors in different postsynaptic neurons in different dStr subregions to convey strikingly different signals.
PMID: 30295607
ISSN: 2050-084x
CID: 3501952

What makes a RAG regeneration associated?

Ma, Thong C; Willis, Dianna E
Regenerative failure remains a significant barrier for functional recovery after central nervous system (CNS) injury. As such, understanding the physiological processes that regulate axon regeneration is a central focus of regenerative medicine. Studying the gene transcription responses to axon injury of regeneration competent neurons, such as those of the peripheral nervous system (PNS), has provided insight into the genes associated with regeneration. Though several individual "regeneration-associated genes" (RAGs) have been identified from these studies, the response to injury likely regulates the expression of functionally coordinated and complementary gene groups. For instance, successful regeneration would require the induction of genes that drive the intrinsic growth capacity of neurons, while simultaneously downregulating the genes that convey environmental inhibitory cues. Thus, this view emphasizes the transcriptional regulation of gene "programs" that contribute to the overall goal of axonal regeneration. Here, we review the known RAGs, focusing on how their transcriptional regulation can reveal the underlying gene programs that drive a regenerative phenotype. Finally, we will discuss paradigms under which we can determine whether these genes are injury-associated, or indeed necessary for regeneration.
PMID: 26300725
ISSN: 1662-5099
CID: 3701902

cAMP-responsive element-binding protein (CREB) and cAMP co-regulate activator protein 1 (AP1)-dependent regeneration-associated gene expression and neurite growth

Ma, Thong C; Barco, Angel; Ratan, Rajiv R; Willis, Dianna E
To regenerate damaged axons, neurons must express a cassette of regeneration-associated genes (RAGs) that increases intrinsic growth capacity and confers resistance to extrinsic inhibitory cues. Here we show that dibutyrl-cAMP or forskolin combined with constitutive-active CREB are superior to either agent alone in driving neurite growth on permissive and inhibitory substrates. Of the RAGs examined, only arginase 1 (Arg1) expression correlated with the increased neurite growth induced by the cAMP/CREB combination, both of which were AP1-dependent. This suggests that cAMP-induced AP1 activity is necessary and interacts with CREB to drive expression of RAGs relevant for regeneration and demonstrates that combining a small molecule (cAMP) with an activated transcription factor (CREB) stimulates the gene expression necessary to enhance axonal regeneration.
PMID: 25296755
ISSN: 1083-351x
CID: 3701892

In vitro ischemia suppresses hypoxic induction of hypoxia-inducible factor-1α by inhibition of synthesis and not enhanced degradation

Karuppagounder, Saravanan S; Basso, Manuela; Sleiman, Sama F; Ma, Thong C; Speer, Rachel E; Smirnova, Natalya A; Gazaryan, Irina G; Ratan, Rajiv R
Hypoxia-inducible factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. This study investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation; OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen-dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to monitor quantitatively distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2-hr hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolylhydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Furthermore, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD vs. hypoxia and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia.
PMID: 23456821
ISSN: 1097-4547
CID: 3701872

Nitration of Hsp90 induces cell death

Franco, Maria Clara; Ye, Yaozu; Refakis, Christian A; Feldman, Jessica L; Stokes, Audrey L; Basso, Manuela; Melero Fernández de Mera, Raquel M; Sparrow, Nicklaus A; Calingasan, Noel Y; Kiaei, Mahmoud; Rhoads, Timothy W; Ma, Thong C; Grumet, Martin; Barnes, Stephen; Beal, M Flint; Beckman, Joseph S; Mehl, Ryan; Estévez, Alvaro G
Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.
PMID: 23487751
ISSN: 1091-6490
CID: 3701882