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Midbrain dopaminergic neurons (mDANs) control voluntary movement, cognition, and reward behavior under physiological conditions and are implicated in human diseases such as Parkinson’s disease (PD). Many transcription factors (TFs) controlling human mDAN differentiation during development have been described, but much of the regulatory landscape remains undefined. Using a tyrosine hydroxylase (TH) iPSC reporter line, we have generated time series transcriptomic and epigenomic profiles of purified mDANs during differentiation. Integrative analysis predicted novel central regulators of mDAN differentiation and super-enhancers were used to prioritize key TFs. We find LBX1, NHLH1 and NR2F1/2 to be necessary for mDAN differentiation and show that overexpression of either LBX1 or NHLH1 can also improve mDAN specification. NHLH1 is necessary for the induction of neuronal miR-124, while LBX1 regulates cholesterol biosynthesis, possibly through mTOR signaling. Consistently, rapamycin treatment led to an inhibition of mDAN differentiation. Thus, our work reveals novel regulators of human mDAN differentiation.

Dopaminergic neurons (DA neurons) are controlled by multiple factors, many involved in neurological disease. Parkinson's disease motor symptoms are caused by the demise of nigral DA neurons, leading to loss of striatal dopamine (DA). Here, we measured DA concentration in the dorsal striatum of 32 members of Collaborative Cross (CC) family and their eight founder strains. Striatal DA varied greatly in founders, and differences were highly heritable in the inbred CC progeny. We identified a locus, containing 164 genes, linked to DA concentration in the dorsal striatum on chromosome X. We used RNAseq profiling of the ventral midbrain of two founders with substantial difference in striatal DA-C56BL/6 J and A/J-to highlight potential protein-coding candidates modulating this trait. Among the five differentially expressed genes within the locus, we found that the gene coding for the collagen IV alpha 6 chain (Col4a6) was expressed nine times less in A/J than in C57BL/6J. Using single cell RNA-seq data from developing human midbrain, we found that COL4A6 is highly expressed in radial glia-like cells and neuronal progenitors, indicating a role in neuronal development. Collagen IV alpha-6 chain (COL4A6) controls axogenesis in simple model organisms. Consistent with these findings, A/J mice had less striatal axonal branching than C57BL/6J mice. We tentatively conclude that DA concentration and axonal branching in dorsal striatum are modulated by COL4A6, possibly during development. Our study shows that genetic mapping based on an easily measured Central Nervous System (CNS) trait, using the CC population, combined with follow-up observations, can parse heritability of such a trait, and nominate novel functions for commonly expressed proteins.

Tau is a microtubule-binding protein that can receive various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Hyperphosphorylation of tau is linked to its aggregation and the formation of neurofibrillary tangles (NFTs), which are a hallmark of Alzheimer’s disease (AD). While more than 70 phosphorylation sites have been detected previously on NFT tau, studies of oligomeric and detergent-soluble tau in human brains during the early stages of AD are lacking. Here we apply a comprehensive electrochemiluminescence ELISA assay to analyze twenty-five different PTM sites as well as tau oligomerization in control and sporadic AD brain. The samples were classified as Braak stages 0–I, II or III–IV, corresponding to the progression of microscopically detectable tau pathology throughout different brain regions. We found that soluble tau multimers are strongly increased at Braak stages III–IV in all brain regions under investigation, including the temporal cortex, which does not contain NFTs or misfolded oligomers at this stage of pathology. We additionally identified five phosphorylation sites that are specifically and consistently increased across the entorhinal cortex, hippocampus and temporal cortex in the same donors. Three of these sites correlate with tau multimerization in all three brain regions, but do not overlap with the epitopes of phospho-sensitive antibodies commonly used for the immunohistochemical detection of NFTs. Our results thus suggest that soluble multimers are characterized by a small set of specific phosphorylation events that differ from those dominating in mature NFTs. These findings shed light on early PTM changes of tau during AD pathogenesis in human brains.

Background: Human tauopathies including Alzheimer's disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance. Methods: We used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons. Results: Our study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130. Conclusions: Our findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon. Funding: Data collection was supported through funding by NIA grants P50 AG016574, R01 AG032990, U01 AG046139, R01 AG018023, U01 AG006576, U01 AG006786, R01 AG025711, R01 AG017216, R01 AG003949, NINDS grant R01 NS080820, CurePSP Foundation, and support from Mayo Foundation. The Brain and Body Donation Program is supported by the National Institute of Neurological Disorders and Stroke (U24 NS072026 National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders), the National Institute on Aging (P30 AG19610 Arizona Alzheimer’s Disease Core Center), the Arizona Department of Health Services (contract 211002, Arizona Alzheimer’s Research Center), the Arizona Biomedical Research Commission (contracts 4001, 0011, 05-901 and 1001 to the Arizona Parkinson’s Disease Consortium) and the Michael J. Fox Foundation for Parkinson’s Research

Human tauopathies including Alzheimer’s disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, leading to the formation of insoluble aggregates, neuronal dysfunction and degeneration. Using a mass spectrometry approach, we identified multiple sites of lysine monomethylation on Tau isolated from a detergent-soluble fraction of human brain, some of which were increased in early AD samples. Brain tissues derived from a mouse model of tauopathy demonstrate an age-dependent increase in methylation at specific sites, with methylated Tau enriched in the soluble nuclear fraction and not associated with hyperphosphorylated, insoluble Tau species. Furthermore, we show that the protein lysine methyltransferase SETD7 methylates Tau at K132 and demonstrate an interaction with K130, an additional methylation site in close vicinity. These findings shed light on the function of a novel type of PTM on Tau that provide a potential signal for its translocation to different subcellular sites. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may furthermore provide insight into this disease-associated phenomenon.

Tau is a microtubule-binding protein that can receive various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Hyperphosphorylation of tau is linked to its aggregation and the formation of neurofibrillary tangles (NFTs), which are a hallmark of Alzheimer’s disease (AD). While more than 70 phosphorylation sites have been detected previously on NFT tau, studies of oligomeric and detergent-soluble tau in human brains during the early stages of AD are lacking. Here we apply a comprehensive electrochemiluminescence ELISA assay to analyze twenty-five different PTM sites as well as tau oligomerization in control and sporadic AD brain. The samples were classified as Braak stages 0-I, II or III-IV, respectively, corresponding to the progression of microscopically detectable tau pathology throughout different brain regions. We find that soluble tau oligomers are strongly increased at Braak stages III-IV in all brain regions under investigation, including the temporal cortex, which does not contain NFTs at this stage of pathology. We additionally identified five phosphorylation sites that are specifically and consistently increased across the entorhinal cortex, hippocampus and temporal cortex in the same donors. Three of these sites correlate with tau oligomerization in all three brain regions, but do not overlap with the epitopes of phospho-sensitive antibodies commonly used for the immunohistochemical detection of NFTs. Our results thus suggest that soluble oligomers are characterized by a small set of specific phosphorylation events that differ from those dominating in mature NFTs and shed light on early PTM changes of tau during AD pathogenesis in human brains.