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  • Intermediate Progenitors Facilitate Intracortical Progression of Thalamocortical Axons and Interneurons through CXCL12 Chemokine Signaling.

    17 October 2018

    Glutamatergic principal neurons, GABAergic interneurons and thalamocortical axons (TCAs) are essential elements of the cerebrocortical network. Principal neurons originate locally from radial glia and intermediate progenitors (IPCs), whereas interneurons and TCAs are of extrinsic origin. Little is known how the assembly of these elements is coordinated. C-X-C motif chemokine 12 (CXCL12), which is known to guide axons outside the neural tube and interneurons in the cortex, is expressed in the meninges and IPCs. Using mouse genetics, we dissected the influence of IPC-derived CXCL12 on TCAs and interneurons by showing that Cxcl12 ablation in IPCs, leaving meningeal Cxcl12 intact, attenuates intracortical TCA growth and disrupts tangential interneuron migration in the subventricular zone. In accordance with strong CXCR4 expression in the forming thalamus and TCAs, we identified a CXCR4-dependent growth-promoting effect of CXCL12 on TCAs in thalamus explants. Together, our findings indicate a cell-autonomous role of CXCR4 in promoting TCA growth. We propose that CXCL12 signals from IPCs link cortical neurogenesis to the progression of TCAs and interneurons spatially and temporally. Significance statement: The cerebral cortex exerts higher brain functions including perceptual and emotional processing. Evolutionary expansion of the mammalian cortex is mediated by intermediate progenitors, transient amplifying cells generating cortical excitatory neurons. During the peak period of cortical neurogenesis, migrating precursors of inhibitory interneurons originating in subcortical areas and thalamic axons invade the cortex. Although defects in the assembly of cortical network elements cause neurological and mental disorders, little is known how neurogenesis, interneuron recruitment, and axonal ingrowth are coordinated. We demonstrate that intermediate progenitors release the chemotactic cytokine CXCL12 to promote intracortical interneuron migration and growth of thalamic axons via the cognate receptor CXCR4. This paracrine signal may ensure thalamocortical connectivity and dispersion of inhibitory neurons in the rapidly growing cortex.

  • Investigating the function of microtubule-associated protein tau (MAPT) and its genetic association with Parkinson’s using human iPSC-derived dopamine neurons

    17 October 2018

    OBJECTIVES The microtubule associated protein tau (MAPT) locus is highly associated with Parkinson's (PD); however, the mechanisms underlying susceptibility remain unclear. We propose that polymorphisms within the MAPT haplotype sequence have functional consequences on MAPT expression and tau protein function in the dopamine neurons notably lost in PD. To examine this we chose the following objectives: • To investigate allele-specific regulation of tau expression in iPSC-derived dopamine neurons. • To investigate the physiological role of tau in axonal transport by perturbing tau expression. METHODS Induced pluripotent stem cells derived from healthy heterozygous (H1/H2) donors were differentiated into midbrain-type neuronal cultures. Dopamine neurons were isolated using rapid fixation immunostaining for tyrosine hydroxylase (TH) followed by fluorescence-activated cell sorting and RNA extraction. For tau perturbation, RNAi targeting specific isoforms or total tau was designed, with initial knockdown performed in neuroblastoma cells. RESULTS Transcripts of TH were >7-fold enriched in FACS-isolated neurons and mature MAPT isoforms (exon 3+ or 10+) were detected with extended culture. siRNA-mediated specific knockdown of exon 3+ (~60%) or exon 10+ (~90%) MAPT isoforms was achieved. Isoform-specific and total MAPT shRNA sequences were incorporated into lentivirus plasmids for delivery to neuronal cultures, in addition to those expressing fluorescent amyloid precursor protein for imaging of live axonal transport. CONCLUSIONS Examining haplotype-specific tau expression and function for the first time in dopamine cultures allows us to understand the effect of haplotype on tau protein in the neurons that degenerate in PD, and to identify therapeutic targets to reduce progression beyond disease’s earliest signs.

  • Investigating the function of microtubule-associated protein tau (MAPT) and its genetic association with Parkinson’s using human iPSC-derived dopamine neurons

    17 October 2018

    OBJECTIVES • To investigate allele-specific regulation of tau expression. • To investigate the physiological role of tau in axonal transport within healthy dopamine neurons. BACKGROUND Microtubule-associated protein tau, encoded by the gene MAPT, is involved in neurodegenerative disease by forming hyperphosphorylated aggregates. However, genome-wide association studies for Parkinson’s implicate common variation at the MAPT locus with Parkinson’s despite a general lack of tau neuropathology. Two common haplotypes exist at the MAPT locus, with haplotype H1 increasing risk for Parkinson’s. Haplotype differences do not affect tau protein sequence, suggesting that risk must lie in altered protein expression. We have previously described haplotype-specific differences in the inclusion of exons 3 and 10 in transcripts from H1 and H2 alleles in post-mortem human brain. METHODS To examine MAPT in the midbrain dopamine neurons notably lost in Parkinson’s, induced pluripotent stem cells derived from healthy heterozygous (H1/H2) donors were differentiated into midbrain-type neuronal cultures. Dopamine neurons were isolated using rapid fixation immunostaining for tyrosine hydroxylase followed by fluorescence-activated cell sorting. RNA was extracted to examine MAPT transcripts on the Sequenom platform. To further investigate regulation of tau expression, candidate microRNAs were expressed in H1/H2 neuroblastoma cells with quantification of tau protein knockdown. The function of tau protein isoforms in axonal transport within human dopamine neurons was examined using live axonal imaging of fluorescent amyloid precursor protein with selective siRNA knockdown of specific tau isoforms or total tau. CONCLUSIONS Understanding how the regulation of MAPT expression can result in altered functionality will help identify therapeutic targets to help prevent Parkinson’s progression from its earliest signs.

  • Progressive dopaminergic alterations and mitochondrial abnormalities in LRRK2 G2019S knock-in mice.

    17 October 2018

    Mutations in the LRRK2 gene represent the most common genetic cause of late onset Parkinson's disease. The physiological and pathological roles of LRRK2 are yet to be fully determined but evidence points towards LRRK2 mutations causing a gain in kinase function, impacting on neuronal maintenance, vesicular dynamics and neurotransmitter release. To explore the role of physiological levels of mutant LRRK2, we created knock-in (KI) mice harboring the most common LRRK2 mutation G2019S in their own genome. We have performed comprehensive dopaminergic, behavioral and neuropathological analyses in this model up to 24months of age. We find elevated kinase activity in the brain of both heterozygous and homozygous mice. Although normal at 6months, by 12months of age, basal and pharmacologically induced extracellular release of dopamine is impaired in both heterozygous and homozygous mice, corroborating previous findings in transgenic models over-expressing mutant LRRK2. Via in vivo microdialysis measurement of basal and drug-evoked extracellular release of dopamine and its metabolites, our findings indicate that exocytotic release from the vesicular pool is impaired. Furthermore, profound mitochondrial abnormalities are evident in the striatum of older homozygous G2019S KI mice, which are consistent with mitochondrial fission arrest. We anticipate that this G2019S mouse line will be a useful pre-clinical model for further evaluation of early mechanistic events in LRRK2 pathogenesis and for second-hit approaches to model disease progression.

  • Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells.

    17 October 2018

    Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes.