Jeffrey D. Macklis - US grants

DESCRIPTION (provided by applicant): The long-term goal of the proposed experiments is the repair of damaged neocortical circuitry. Much of our prior work has focused on repair by transplantation of immature neurons and neural precursors. Recently, however, we have manipulated endogenous precursors in situ in the adult mouse to undergo neurogenesis and anatomic circuit re-formation de novo in the neocortex, where it does not normally occur, This was without transplantation. This work aims toward the ultimate goal of repair by manipulation of endogenous neural precursors in situ. This could lead to therapies for degenerative, developmental, or acquired diseases of cortex and its output circuitry (e.g. spinal cord). In neocortex, the effectiveness of such future therapies could depend critically on whether endogenous precursors, or stem cells, can be precisely induced to form new neurons; migrate to correct locations; differentiate and integrate appropriately; and re-form precise long-distance projections and complex functional connections. Neuroblasts and neural precursors respond specifically to altered expression of local signal molecules in regions of cortex undergoing synchronous biophysically-induced apoptosis of projection neurons. They selectively migrate into such regions, differentiate into projection neurons, receive synaptic input, and re-form long-distance circuitry. Though we have made considerable progress in identifying conditions under which cortical neurogenesis and partial repair of cortical circuitry is possible in the adult, many questions still remain to be investigated. These questions form the basis of the proposed research: 1) Can we substantially increase the number of new cortical neurons by manipulating proliferation/differentiation of endogenous precursors &/or survival of newborn neurons? 2) Can newborn neurons differentiate precisely into new functional projection-neurons, receive afferent synapses, and become functionally integrated? 3) What are the molecular mechanisms responsible for inducing neurogenesis and specific differentiation by endogenous precursors in the adult neocortex? Our three specific aims will test and investigate these questions directly. Proposed experiments will: Aim 1) determine the effects of select candidate growth factors toward increasing the amount of induced neurogenesis in neocortex of adult mice; Aim 2) investigate the precision of newborn neuron differentiation by analysis of neurotransmitter and receptor complement and synaptic integration, using confocal and immunocytochemistry; and Aim 3) investigate the molecular mechanisms of this induced neurogenesis via microarray analysis of differential gene expression, confirmation of candidates, and in vitro functional assay. Together, these experiments will investigate the molecular mechanisms underlying induced neurogenesis in the adult murine neocortex; the potential for substantially increasing the amount of neurogenesis; and the ability of newborn cortical neurons derived from endogenous precursors to repair cortical circuitry.

DESCRIPTION (provided by applicant): The long-term goal of the proposed experiments is the repair of damaged or degenerated cortico-spinal circuitry via i) induction of neurogenesis of new cortico-spinal motor neurons (CSMN) from endogenous precursors ("stem cells"); ii) support of their survival, and iii) recruitment into functional synaptic circuitry with the lower motor neuron population. Recently, we have manipulated endogenous precursors in situ in the adult mouse to undergo neurogenesis (birth of new neurons) de novo in adult mouse neocortex, where it does not normally occur. In collaborative work, we induced behaviorally functional neuronal replacement in situ in songbirds by homologous avian endogenous precursors. These experiments demonstrated that there exists a sequence and combination of molecular signals by which the birth of new neurons can be induced, even in the adult neocortex where neurogenesis does not normally occur. Even more directly relevant to this proposal are our recent findings that 1) endogenous precursors can also be specifically induced to differentiate in situ into cortico-spinal motor neurons (CSMN) with projections to the spinal cord; and 2) that we can isolate, FACS purify (to >99.5% purity), and culture CSMN at distinct developmental stages for analysis of lineage-specific controls over survival and differentiation, particularly rate / extent of axon elongation. We hypothesize that we can substantially increase the number of newly recruited CSMN by enhancing the survival of the newborn neurons and/or their rate and extent of axonal outgrowth to supportive spinal cord targets. We also hypothesize that newborn neurons can differentiate precisely into new functional CSMN, receive afferent synapses, and become synaptically integrated. Our five Aims will test these and related hypotheses directly, in vitro and in vivo. The proposed research will: Aim 1) undertake a more complete analysis of lineage-specific differentiation of the induced adult-born CSMN, and a more detailed characterization of the time course of induced neurogenesis, neuronal differentiation, connectivity, and long-term survival of newborn CSMN); Aims 2,3) investigate in vitro a select set of candidate growth and neurotrophic factors enabling potentially stage-specific enhanced survival and/or enhanced outgrowth and circuit connectivity of CSMN, employing FACS purification of CSMN at distinct developmental stages, and bath vs. localized factor application; Aim 4) manipulate and increase the induced adult CSMN neurogenesis and spinal projections by intraventricular infusion of highly selected candidate growth factors from the in vitro experiments and Aim 5) investigate the precision of CSMN differentiation and potential afferent and efferent synapse formation using ICC and retroviral GFP-barley lectin expression. Together, this work aims toward the ultimate goal of repair of cortico-spinal motor neuron circuitry by manipulation of endogenous neural precursors in situ, without transplantation.

DESCRIPTION (provided by applicant): The long-term goal of the proposed experiments is the repair of damaged neocortical circuitry. The current work is focused on repair by transplantation of immature neurons. Ultimately, however, repair may also be possible by manipulation of endogenous neural precursors without transplantation. These approaches could lead to therapies for degenerative, developmental, or acquired diseases of neocortex. The effectiveness of such future therapies will depend critically on whether new neurons 1) migrate to correct locations, 2) differentiate precisely, 3) form correct long-distance projections, and 4) function within circuitry. Prior work of this grant program has focused on migration, differentiation, and connectivity. This proposal focuses on functional circuit integration. We have previously shown that transplanted immature neurons and multipotent neural precursors can selectively migrate into regions of adult mouse cortex undergoing targeted neurodegeneration of CPN, differentiate into projection-neurons, accept synaptic input, and re-form axonal projections. Experiments of the prior grant period reveal that later stage immature neurons repair circuitry with higher efficiency than earlier stage precursors. Recently, we have also manipulated endogenous precursors in situ in the adult mouse to undergo neurogenesis- the birth of new neurons- and re-formation of cortico-thalamic or cortico-spinal circuitry de novo in the adult mouse neocortex, where it does not normally occur89. This recruitment was without transplantation. A central and critical issue in the field is whether newly incorporated neurons become functionally integrated within CNS circuitry, and whether they can actually contribute to function and behavior. We have developed a range of molecular, electrophysiological, and functional sensory activation approaches to rigorously investigate to what extent the anatomic, morphologic, and synaptic integration of transplanted neurons we have previously demonstrated will be reflected in functional circuit integration, using eGFP+ donor neurons for detailed analysis of connectivity. We will also apply new approaches to enhance the cellular integration and the functional circuit formation by newly incorporated neurons. Our four specific aims will investigate whether new neurons in adult vibrissal somatosensory cortex Aim 1) acquire the precise phenotype of endogenous neurons in their cellular and temporal patterns of activation-dependent molecular signaling events;Aim 2) acquire the precise electrophysiologic phenotype to become functionally activated by sensory input;Aim 3) can be enhanced in their circuit integration by intracortical infusion of rationally selected peptide growth factors;Aim 4) can undergo enhanced functional circuit integration via enriched environmental sensory stimulation of the relevant circuitry.

DESCRIPTION (provided by applicant): The long-term goal of the proposed experiments is repair of neocortical projection neuron circuitry. This work aims toward the ultimate goal of repair by manipulation of endogenous neural progenitors in situ. This could lead to therapies for degenerative, developmental, or acquired diseases of cortex and its output circuitry (e.g. corticospinal). In neocortex, the effectiveness of such future therapies could depend critically on whether endogenous progenitors can be precisely induced to form the correct, subtype-specific neurons; differentiate and integrate appropriately; and re-form long-distance projections and complex functional connections. At the time of submission for the initial period of this grant, we had recently published (Magavi, Nature, 2000; Scharff, Neuron, 2000) the field's first demonstrations of induction of neurogenesis, the birth of new neurons, from endogenous progenitors in the adult brain. We chose corticothalamic projection neurons (CThPN) and their development for focused study in mice toward induction of neurogenesis because they are a prototypical population of long-distance cortical projection neurons, and because of their location closest to the available pool of caudal cortical SVZ progenitors. We hypothesized (now with substantial data during development and pilot adult data) that there exist partially fate-specified neocortical progenitors competent to differentiate into corticofugal neurons, including CThPN (Molyneaux, Neuron, 2005; Arlotta, Neuron, 2005; Molyneaux, Nat Rev NSci, 2007; Lai, Neuron, 2008; Joshi, Neuron, 2008; Azim, 2008). A next logical step toward future therapeutic manipulation of endogenous progenitors and induction of neurogenesis will be directed differentiation of specific neuron populations by manipulating combinatorial molecular-genetic controls. Though we have made considerable progress identifying cellular and molecular conditions that enable cortical neurogenesis and partial repair of adult cortical circuitry, many questions still remain to be investigated. These questions form the basis of the proposed research. Building on recent results, proposed experiments (Aim 1) functionally investigate FOG-2, a newly identified transcriptional regulator critical for CThPN development, using loss- and gain-of-function in vivo; (Aim 2) investigate two new candidate combinatorial molecular-genetic controls over CThPN birth and development; (Aim 3) investigate whether partially fate- restricted neural progenitors, recently identified during development, exist in the adult mouse neocortex, with potentially enhanced competence to generate corticofugal neurons; and (Aim 4) induce CThPN neurogenesis from (potentially) partially fate-restricted progenitors in the adult mouse forebrain via manipulation of critical molecular-genetic controls over CThPN development. Together, these experiments will significantly advance our ability to induce type-specific neurogenesis and ultimately direct functional circuit repair of the adult CNS.

7. PROJECT SUMMARY / ABSTRACT The long-term goals of the proposed experiments are both to elucidate molecular-genetic controls over the neuron subtype-specific development of corticospinal motor neurons (CSMN) (and related neocortical projection neurons), and to potentially enable future approaches to repair of degenerating or injured CSMN. CSMN are both developmentally prototypical for all neocortical projection neurons, and clinically important as the brain neurons that degenerate in amyotrophic lateral sclerosis / motor neuron disease (ALS/MND) and whose axonal injury is central to loss of motor function in spinal cord injury. Proposed experiments will deeply investigate function of the centrally important CSMN/subcerebral-specific transcription factor CTIP2 (COUP-TF interacting protein 2) and its paralog CTIP1 in development of CSMN and related neurons in murine neocortex. Ctip2 has increasingly emerged as both a critical regulator of development and connectivity of CSMN, and as a common target for regulation (largely repression) by multiple projection neuron subtype differentiation pathways. Ctip2 is known from other organ systems to be involved in developmental lineage specification decisions. Within the neocortex, CTIP2 is specifically expressed by CSMN and related subcerebral projection neurons, and is necessary for outgrowth, fasciculation, and targeting of CSMN axons. While Ctip2 has emerged as centrally important for CSMN development, most aspects of its function remain unknown. Substantial preliminary data support these aims. Previous work from this laboratory identified Ctip2 as a critical CSMN molecular control, and demonstrated that CSMN axons in Ctip2-/- mice are misrouted before penetrating the internal capsule (IC), defasciculate in the IC, and fail to project to the spinal cord (SC). Because CTIP2 also controls differentiation of striatal medium-sized spiny neurons (MSN), which surround CSMN axons in the IC, the hypothesis is suggested that some defects in Ctip2-/- CSMN connectivity to SC might result from dysregulation of axon growth and guidance controls in Ctip2-/- MSN. Mice lacking Ctip2 only in neocortex (Emx1-Cre;Ctip2fl/fl) reveal that a subset of CSMN enter and fasciculate in the IC, and some even reach the SC. Other preliminary studies find that the Ctip2 paralog Ctip1 interacts cross-repressively with Ctip2 to control deep-layer projection neuron development, and that Ctip1 additionally regulates areal organization. Proposed experiments will: (Aims 1, 2) delineate CSMN-autonomous and non-CSMN-autonomous roles of Ctip2 in CSMN axon growth and fasciculation; (Aims 3, 4) investigate a newly-identified genetically cross- repressive interaction between Ctip2 and its paralog Ctip1 in CSMN development, as well as independent roles of Ctip1 in areal organization and development of other deep-layer projection neurons. Experiments beyond this proposal could identify genes regulated directly or indirectly by Ctip2 in CSMN. These studies will elucidate mechanisms by which Ctip2, a central regulator of CSMN differentiation, acts alone and with other genes to instruct the precision of development of this developmentally prototypical, clinically important neuron type.

The major goal of the proposed research is to elucidate central molecular controls and regulatory mechanisms critical for development, identity, and subtype diversity of neocortical corticofugal (output) projection neurons (CFuPN), which might also enable their future directed differentiation, or molecular-cellular manipulation / ?reprogramming?. CFuPN are the broad population of cerebral cortex excitatory output neurons that extend axons away from cortex to subcortical targets. Dysregulation of CFuPN developmental controls contributes to human developmental diseases? and potentially later to selective vulnerability in neurodegenerative diseases. CFuPN include corticospinal motor neurons (CSMN), the broader subpopulation of subcerebral projection neurons (SCPN), corticothalamic projection neurons (CThPN), and major subsets of corticostriatal projection neurons (CStrPN). They play key roles in motor, sensory, high-level cognitive, and behavioral functions. Distinct CFuPN subtypes are specifically vulnerable and central to distinct disorders. CSMN/SCPN degenerate with spinal motor neurons in ALS; CStrPN with striatal MSN in Huntington's disease. CSMN damage is central in spinal cord injury. Cerebral palsy involves CFuPN injuries; some pain syndromes and epilepsies, CThPN. Understanding controls over development and maintenance of CFuPN diversity and precision will contribute centrally to basic understanding of the complex organization and function of cortex and its output circuitry, to identification of causes and therapeutic approaches for disease involving specific CFuPN subtypes, and to future strategies for cortical disease modeling, therapeutic screening, and regeneration/repair, since manipulation of these controls might also enable directed differentiation of pluripotent stem cells (m/h ES, iPS) for accurate disease models for mechanistic study and therapy discovery; directed differentiation of appropriate progenitors for regeneration; and potentially ?reprogramming? of some CFuPN (e.g., a subset of CThPN) into clinically desired subtypes (e.g., CSMN). We and others have made substantial progress identifying some broad, overall molecular/transcriptional controls over CFuPN subtype development, but much remains to be discovered at the subtype-specific level most relevant for disease and understanding of cortical function and organization. Building on our previous work, we propose to investigate novel regulatory biology of CFuPN subtype differentiation, diversity, and maintenance, substantially focused through a `lens' of regulation by Tle4 of identity acquisition of the distinct and clinically relevant CThPN, CSMN, and related SCPN subtypes (Aim 1), and to identify core transcriptional mechanisms at the molecular level that enable this precise regulation of CFuPN subtype diversity and balance (Aim 2). Further, based on highly motivating pilot studies indicating that Tle4 function is required to maintain CThPN vs. CSMN distinction even after circuit formation, we will rigorously and deeply investigate the potential to interconvert or ?reprogram? CFuPN subtype identity of some CThPN into ?genuine? CSMN by manipulation of Tle4 function at progressively later stages of development (Aim 3), exploring limits of subtype plasticity and circuitry manipulation.  

The long-term goals of the proposed research are both to elucidate central molecular controls over development and diversity of neocortical callosal projection neuron (CPN) connectivity, and to identify potential causes and therapeutic approaches to disease involving CPN circuitry. CPN are the broad population of inter-hemispheric pyramidal neurons whose axons connect the two cerebral hemispheres via the corpus callosum. CPN play key roles in high-level associative, integrative, cognitive, behavioral, sensory, and motor functions, based on precise, area-specific CPN subtype connectivity and diversity. Disruptions in CPN development are correlated with deficits in multiple disorders, including agenesis of the corpus callosum, autism spectrum disorders, and schizophrenia. Currently, how the remarkable diversity of CPN subtypes and connectivity is specified, and how transcriptional programs implement specific connectivity via local, cell-autonomous effectors, is unknown. Our lab recently identified a combinatorially-expressed set of genes that both define CPN as a broad population, and identify novel subpopulations of CPN during development (Neuron, 2005, 2016a,b; J Neurosci, 2009; Cer Cor, 2016a,b). We also developed innovative approaches to investigate subtype-specific, subcellular growth cone (GC) molecular machinery. Building on this work, we propose deep and rigorous functional investigation of Cited2 control over precise CPN connectivity & circuit wiring, including RNAs & proteins detected uniquely in GCs. Cited2 is an exemplar transcriptional co-regulator that we hypothesize functions importantly in development of precise areally- and functionally-specific CPN circuitry in somatosensory cortex, and its dysfunction elucidates disorders of CPN connectivity and diversity. We have already identified that Cited2 regulates and refines two stages of precise CPN development and diversity, functioning 1) broadly in basal progenitors to regulate generation of superficial layer CPN, and 2) postmitotically in an area-restricted manner to refine distinct, precise identity and development of somatosensory (S1) CPN. To connect Cited2 transcriptional regulation to local implementation of S1 CPN connectivity in developing GCs, we propose to: Aim 1) investigate CPN-autonomy of Cited2 regulatory function in S1 CPN postmitotic development and connectivity, via novel mosaic, recombinase-based genetic manipulation technology (?BEAM?) for dual population analysis; Aim 2) investigate GC & soma RNA & proteomes of WT vs Cited2 cKO S1 CPN during axon development via new and innovative approaches, to gain direct mechanistic understanding of CPN circuit development at critical developmental stages; Aim 3) investigate the specific function of GC-localized downstream effectors that are dysregulated in Cited2-null CPN; and Aim 4) investigate the integrated function of precise CPN circuit development in cognitive & ASD-relevant behavior. Together, the proposed studies will provide substantial insight from gene to circuit to behavior into molecular control over development, diversity, and precision of connectivity of CPN subtypes with distinct function and integration of cortical information, processes centrally disrupted in human disorders. Controls over CPN connectivity are now essentially unknown, and transcriptional dysregulation has not been previously connected to downstream local effectors of circuit development. This research will contribute to understanding cortical organization, function, and potentially toward prevention, diagnosis, and therapy of human disorders.

An overarching, central question in all of neuroscience is the specificity, modification, and function of the immense diversity of function-specific circuitry? a question still inaccessible in multiple core aspects. This is what underlies how the brain-nervous system senses, integrates, moves the body, thinks, functions with precision, malfunctions with specificity in disease, degenerates with circuit specificity, might be regenerated, and/or might be modeled in culture. What actually implements and maintains circuit specificity is a key, core issue from developmental specificity of circuits, to developmental abnormalities, to proper function (or dysfunction) and circuit type-specific molecular regulators, to subtype-specific degeneration (e.g. in ALS, Huntington's, Parkinson's diseases), to regeneration (or typical lack thereof) in the CNS for spinal cord injury or with optimal accuracy in the PNS, to mechanistic and therapeutic modeling of disease using iPS/ES-derived neurons. Growth cones (GCs) ?build? circuits and mature into synapses, where human genomic risk associations are showing up in neuropsychiatric diseases such as schizophrenia, autism, bipolar disorder, developmental intellectual disabilities. I propose uniquely enabling, pioneering work on these issues? now possible by our innovative approaches. We are now able to directly investigate molecular machinery of distinct GC subtypes, thus distinct circuits. Despite their importance, we know little about the diversity and specialization of circuit-specific GCs? the subcellular molecular machines that implement specific circuit wiring, mature with precision into presynaptic halves of immensely diverse synapses, and control the long-standing ?sorting problem?. GCs perform these functions over many days of development for each pathway, often 103-105 cell body diameters away from the nucleus and transcriptional control. Remarkably, but rarely considered, one nucleus, with one transcriptional regulatory machinery, can control 2 or more divergent GCs to wire multi-target circuitry. I propose entirely new, highly innovative, pioneering work in development, cell biology, disease, and regeneration (also relevant to modeling) to uniquely address this critical gap in knowledge. We developed new approaches to investigate subtype- and stage-specific GCs directly from brains, with high-depth, quantitative proteomic and RNA analysis, and have already completed proof-of-concept experiments enabling a range of pioneering new work. We selectively purify GCs based on neuron subtype, projection trajectory, and developmental stage using a combination of molecular, anatomic, and genetic labeling strategies; subcellular biochemistry; newly developed small-particle sorting; peptide mass spectrometry; and Next Gen sequencing. Simultaneous isolation of protein and RNA from parent somas and their GCs identifies hundreds of proteins and transcripts enriched orders of magnitude in GCs, essentially not detected in parent somas. This indicates that investigation of GCs might actually be required to understand subtype-specific circuitry. GCs appear to be ?programmed? early, then ?poised? to exert quite autonomous local control. I propose ambitious and venturesome investigations of subtype-, stage-, and target-specific GC proteins and RNAs in multiple specific settings to study mechanisms of development, cell biology, disease, regeneration, & iPS/ES models. These directions range from immediate, to ~5 yrs, to an ~10 yr horizon. Results will generate new hypotheses and investigations.

To enable powerful genetic mosaic investigation of gene function in complex systems? without current limitations of gene location or toxicity? in particular for development and diversity of neuronal subtypes and their complex circuitry in cerebral cortex, we propose to develop and implement two entirely novel, innovative, inter-related systems for genetic mosaic functional analysis. These systems enable binary? all-or-none, neuron-by-neuron ?aleatory?? random, mosaic analysis with control over ratios of wt and genetically manipulated cells. The proposed work will substantially extend the range of tools available for mosaic analysis. We first propose to further develop and adapt for AAV viral use a new plasmid-based transfection system, BEAM (for binary expression aleatory mosaic), which relies on sparse recombinase activation to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Since all requisite plasmids can be delivered by electroporation or viral transduction, BEAM can be used directly on wild-type or floxed mice, without the need for complex breeding schemes. We also propose to engineer a Rosa26 reporter allele in mice, R26BEACON (for binary expression aleatory cre-operated nested mosaic), which will use Flp to stochastically recombine incompatible frt-site variants, thereby generating green cells that express Cre (EGFP-positive;Cre-positive) and red cells that do not express Cre (tdTomato-positive;Cre-negative). Because green cells express Cre, R26BEACON can be used to delete genes of interest using existing floxed alleles, and to activate expression of effector molecules using existing Rosa26 alleles for cell ablation, modulation of membrane potential, and transcellular labeling. In addition, unlike existing systems of MADM and MASTR, which can be activated only sparsely, R26BEACON will be able to be activated efficiently throughout the entire organism, or in a specific organ or cell type of interest. The motivating biological goals of the proposed work are both to elucidate central molecular controls and regulatory mechanisms over development, subtype diversity, circuit formation, and potential regeneration of cortical projection neurons (PN), and to identify potential causes and therapeutic approaches to dysgenesis and disease involving PN. These innovative BEAM and R26BEACON mosaic systems will uniquely enable new discovery in multiple fields. Toward Aim 1, we have completed highly motivating studies developing BEAM as an already successful approach for electroporation. We now propose to further develop and validate the BEAM plasmid system, and to generate an AAV virus-based BEAM system, 1) testing fidelity of reporters and recombination status, 2) analyzing cell autonomy, and 3) generating an AAV-based BEAM. Toward Aim 2, we have already generated a R26BEACON targeting construct by placing an operator module in tandem with a reporter module, which labels cells red in the presence of Flp, and green in either the presence of Cre alone or Cre and Flp in combination. We propose to 1) generate R26BEACON mice, and 2) validate with floxed alleles. BEAM and R26BEACON are novel and innovative. This work advances the tradition of cutting edge recombinase-based genetic approaches such as MADM, Brainbow, and other genetic analysis tools generated by the neuroscience community, enabling investigation of CNS complexity and diversity. Our deep investigation of PN development, diversity, and connectivity provide the foundation to innovatively and rigorously develop and implement BEAM and R26BEACON.