Doctor of Philosophy, University of Pennsylvania (2010)
Bachelor of Science, University of California Santa Barbara (2004)
Thomas Sudhof, Postdoctoral Research Mentor
Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.
View details for DOI 10.1016/j.neuron.2013.05.029
View details for Web of Science ID 000320743400006
Mutations in ATP13A2, which encodes a lysosomal P-type ATPase of unknown function, cause an autosomal recessive parkinsonian syndrome. With mammalian cells, we show that ATP13A2 expression protects against manganese and nickel toxicity, in addition to proteasomal, mitochondrial, and oxidative stress. Consistent with a recessive mode of inheritance of gene defects, disease-causing mutations F182L and G504R are prone to misfolding and do not protect against manganese and nickel toxicity because they are unstable as a result of degradation via the endoplasmic reticulum-associated degradation (ERAD)-proteasome system. The protective effects of ATP13A2 expression are not due to inhibition of apoptotic pathways or a reduction in typical stress pathways, insofar as these pathways are still activated in challenged ATP13A2-expressing cells; however, these cells display a dramatic reduction in the accumulation of oxidized and damaged proteins. These data indicate that, contrary to a previous suggestion, ATP13A2 is unlikely to convey cellular resilience simply by acting as a lysosomal manganese transporter. Consistent with the recent identification of an ATP13A2 recessive mutation in Tibetan terriers that develop neurodegeneration with neuronal ceroid lipofucinoses, our data suggest that ATP13A2 may function to import a cofactor required for the function of a lysosome enzyme(s).
View details for DOI 10.1002/jnr.23112
View details for Web of Science ID 000310337300008
View details for PubMedID 22847264
Synucleinopathies are a group of neurodegenerative disorders, including Parkinson disease, associated with neuronal amyloid inclusions comprised of the presynaptic protein ?-synuclein (?-syn); however the biological events that initiate and lead to the formation of these inclusions are still poorly understood. There is mounting evidence that intracellular ?-syn aggregation may proceed via a seeding mechanism and could spread between neurons through a prion-like mechanism that may involve other amyloidogenic proteins. Several lines of evidence suggest that A? peptides and/or extracellular A? deposits may directly or indirectly promote intracellular ?-syn aggregation. To assess the effects of A? peptides and extracellular A? deposits on ?-syn aggregate formation, transgenic mice (line M83) expressing A53T human ?-syn that are sensitive to developing ?-syn pathological inclusions were cross bred to Tg2576 transgenic mice that generated elevated levels of A? peptides and develop abundant A? plaques. In addition these mice were bred to mice with the P264L presenilin-1 knock-in mutation that further promotes A? plaque formation. These mice demonstrated the expected formation of A? plaques; however despite the accumulation of hyperphosphorylated ?-syn dystrophic neurites within or surrounding A? plaques, no additional ?-syn pathologies were observed. These studies show that A? amyloid deposits can cause the local aggregation of ?-syn, but these did not lead to more extensive ?-syn pathology.
View details for DOI 10.1016/j.neulet.2011.12.005
View details for Web of Science ID 000301695900010
View details for PubMedID 22188655
Synucleinopathies are a group of neurodegenerative disorders associated with the formation of aberrant amyloid inclusions composed of the normally soluble presynaptic protein ?-synuclein (?-syn). Parkinson disease is the most well known of these disorders because it bears ?-syn pathological inclusions known as Lewy bodies (LBs). Mutations in the gene for ?-syn, including the E46K missense mutation, are sufficient to cause Parkinson disease as well as other synucleinopathies like dementia with LBs. Herein, we describe transgenic mice expressing E46K human ?-syn in CNS neurons that develop detrimental age-dependent motor impairments. These animals accumulate age-dependent intracytoplasmic neuronal ?-syn inclusions that parallel disease and recapitulate the biochemical, histological, and morphological properties of LBs. Surprisingly, the morphology of ?-syn inclusions in E46K human ?-syn transgenic mice more closely resemble LBs than the previously described transgenic mice (line M83) that express neuronal A53T human ?-syn. E46K human ?-syn mice also develop abundant neuronal tau inclusions that resemble neurofibrillary tangles. Subsequent studies on the ability of E46K ?-syn to induce tau inclusions in cellular models suggest that both direct and indirect mechanisms of protein aggregation are probably involved in the formation of the tau inclusions observed here in vivo. Re-evaluation of presymptomatic transgenic mice expressing A53T human ?-syn reveals that the formation of ?-syn inclusions in mice must be synchronized; however, inclusion formation is diffuse within affected areas of the neuroaxis such that there was no clustering of inclusions. Collectively, these findings provide insights in the mechanisms of formation of these aberrant proteinaceous inclusions and support the notion that ?-syn aggregates are involved in the pathogenesis of human diseases.
View details for DOI 10.1074/jbc.M111.247965
View details for Web of Science ID 000295406300064
View details for PubMedID 21846727
Parkinson disease (PD) is the most common movement disorder. It is characterized by bradykinesia, postural instability, resting tremor, and rigidity associated with the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Another pathological hallmark of PD is the presence of ?-synuclein proteiniacous inclusions, known as Lewy bodies and Lewy neurites, in some of the remaining dopaminergic neurons. Mounting evidence indicates that both genetic and environmental factors contribute to the etiology of PD. For example, genetic mutations (duplications, triplications or missense mutations) in the ?-synuclein gene can lead to PD, but even in these patients, age-dependent physiological changes or environmental exposures appear to be involved in disease presentation. Several additional alterations in many other genes have been established to either cause or increase the risk of parkinson disease. More specifically, autosomal dominant missense mutations in the gene for leucine-rich repeat kinase 2 (LRRK2/PARK8) are the most common known cause of PD. Recently it was shown that G2019S, the most common diseasing-causing mutant of LRRK2, has dramatic effects on the kinase activity of LRRK2: while activity of wild-type LRRK2 is inhibited by manganese, the G2019S mutation abrogates this inhibition. Based on the in vitro kinetic properties of LRRK2 in the presence of manganese, we proposed that LRRK2 may be a sensor of cytoplasmic manganese levels and that the G2019S mutant has lost this function. This finding, alongside a growing number of studies demonstrating an interaction between PD-associated proteins and manganese, suggest that dysregulation of neuronal manganese homeostasis over a lifetime can play an important role in the etiology of PD.
View details for DOI 10.1016/j.neuro.2011.01.003
View details for Web of Science ID 000296671800015
View details for PubMedID 21238487
Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common cause of late-onset Parkinson disease. Previously, we showed that the G2019S pathogenic mutation can cause a dramatic increase (approximately 10-fold) in kinase activity, far above other published studies. A notable experimental difference was the use of Mn-ATP as a substrate. Therefore, the effects of metal cation-ATP cofactors on LRRK2 kinase activity were investigated. It is shown, using several divalent metal cations, that only Mg(2+) or Mn(2+) can support LRRK2 kinase activity. However, for wild-type, I2020T, and R1441C LRRK2, Mn(2+) was significantly less effective at supporting kinase activity. In sharp contrast, both Mn(2+) and Mg(2+) were effective at supporting the activity of G2019S LRRK2. These divergent effects associated with divalent cation usage and the G2019S mutation were predominantly because of differences in catalytic rates. However, LRRK2 was shown to have much lower (approximately 40-fold) ATP K(m) for Mn-ATP compared with Mg-ATP. Consequently, sub-stoichiometric concentrations of Mn(2+) can act to inhibit the kinase activity of wild-type, but not G2019S LRRK2 in the presence of Mg(2+) . From these findings, a new model is proposed for a possible function of LRRK2 and the consequence of the G2019S LRRK2 pathogenic mutation.
View details for DOI 10.1111/j.1471-4159.2010.06894.x
View details for Web of Science ID 000281828200004
View details for PubMedID 20626563
Mutations in leucine-rich repeat kinase-2 (LRRK2) are the most common known cause of Parkinson disease, but how this protein results in the pathobiology of Parkinson disease is unknown. Moreover, there is variability in pathology among cases, and alpha-synuclein (alpha-syn) neuronal inclusions are often present, but whether LRRK2 is present in these pathological inclusions is controversial. This study characterizes novel LRRK2 antibodies, some of which preferentially recognize an aggregated form of LRRK2, as observed in cell culture models. Large perinuclear aggregates containing LRRK2 were promoted by proteasome inhibition and prevented by microtubule polymerization inhibition. Furthermore, they were vimentin- and gamma-tubulin- but not lamp1-immunoreactive, suggesting that these structures fit the definition of aggresomes. Inhibition of heat shock protein 90 led to the degradation of only the soluble/cytosolic pool of LRRK2, suggesting that the aggresomes formed independent of the stability provided by the heat shock protein 90. Although these novel anti-LRRK2 antibodies identified aggregates in model cell systems, they did not immunostain pathological inclusions in human brains. Furthermore, coexpression of LRRK2 and alpha-syn did not recruit alpha-syn into aggresomes in cultured cells, even in the presence of proteasome inhibition. Thus, although LRRK2 is a model system for aggresome formation, LRRK2 is not present in alpha-syn pathological inclusions.
View details for Web of Science ID 000267557400007
View details for PubMedID 19535993
Mutations in leucine-repeat rich kinase 2 (LRRK2) are the most common known cause of late-onset Parkinson's disease. In this study, a novel system to purify active recombinant LRRK2 expressed in mammalian cells was generated. This recombinant enzyme was used to characterize the specificity of LRRK2 and identify small compounds that can inhibit the kinase activity. Recombinant LRRK2 was shown to autophosphorylate and phosphorylate MBP and a peptide (LRRKtide) corresponding to the T558 [corrected] site in moesin. A series of well-characterized kinase peptide substrates was not modified by LRRK2 demonstrating remarkable specificity. G2019S, the most common disease-causing mutation in LRRK2, increased kinase activity more dramatically than previously appreciated ( approximately 10-fold). Several small molecules sharing a basic indolocarbazole structure (Gö6976, K-252a, and staurosporine) where identified as potent inhibitors of LRRK2 kinase activity. These findings provide important insights and tools to study the mechanisms of LRRK2 pathobiology, and could lead to therapeutic applications.
View details for DOI 10.1016/j.bbrc.2008.11.048
View details for Web of Science ID 000262343200029
View details for PubMedID 19027715
Mutations in LRRK2 are the single most common known cause of Parkinson's disease (PD). Two new PD patients with LRRK2 mutation were identified from a cohort with extensive postmortem assessment. One of these patients harbors the R793M mutation and presented with the typical clinical and pathological features of PD. A novel L1165P mutation was identified in a second patient. This patient had the classical and pathological features of PD, but additionally developed severe neuropsychological symptoms and dementia associated with abundant neurofibrillary tangles in the hippocampal formation; features consistent with a secondary diagnosis of tangle-predominant dementia. alpha-Synuclein-containing pathological inclusions in these patients also were highly phosphorylated at Ser-129, similar to other patients with idiopathic PD. These two PD patients also were characterized by the presence of occasional cytoplasmic TDP-43 inclusions in the temporal cortex, a finding that was not observed in three other patients with the G2019S mutation in LRRK2. These findings extend the clinical and pathological features that may be associated with LRRK2 mutations.
View details for DOI 10.1002/mds.22096
View details for Web of Science ID 000262992800004
View details for PubMedID 19006185
Mutations in leucine-rich repeat kinase 2 (LRRK2) recently have been identified as the most common genetic cause of late-onset sporadic and familial Parkinson's disease (PD). The studies herein explore the biological and pathological properties of Lrrk2.Genetic analysis was performed to identify autopsied patients with the most common Lrrk2 mutation (G2019S). Using an antibody specific to Lrrk2, the biochemical and immunocytochemical distribution of Lrrk2 was assessed.Three patients with the G2019S Lrrk2 mutation were identified. Two patients demonstrated classic PD with Lewy bodies, although concurrent pathological changes consistent with Alzheimer's disease were also present in one of these individuals. The third patient was characterized by parkinsonism without Lewy bodies but demonstrated dystrophic neurites in the substantia nigra intensely stained for Lrrk2. Lrrk2 accumulations were unique to this patient and Lrrk2 was not detected in other types of pathological inclusions. Biochemical analysis showed that Lrrk2 is predominantly a soluble approximately 250 kDa cytoplasmic protein expressed throughout the brain but also in many other organs.The reasons for the selective predisposition of patients with mutations in LRRK2 to develop parkinsonism remains unclear, but Lrrk2 mutations may prime select neuronal populations to cellular insults that can lead to aberrant protein aggregation.
View details for DOI 10.1002/ana.20791
View details for Web of Science ID 000235224400012
View details for PubMedID 16437584