Neuronal TIMP2 regulates hippocampus-dependent plasticity and extracellular matrix complexity.

Functional output of the hippocampus, a brain region subserving memory function, depends on highly orchestrated cellular and molecular processes that regulate synaptic plasticity throughout life. The structural requirements of such plasticity and molecular events involved in this regulation are poorly understood. Specific molecules, including tissue inhibitor of metalloproteinases-2 (TIMP2) have been implicated in plasticity processes in the hippocampus, a role that decreases with brain aging as expression is lost. Here, we report that TIMP2 is highly expressed by neurons within the hippocampus and its loss drives changes in cellular programs related to adult neurogenesis and dendritic spine turnover with corresponding impairments in hippocampus-dependent memory. Consistent with the accumulation of extracellular matrix (ECM) in the hippocampus we observe with aging, we find that TIMP2 acts to reduce accumulation of ECM around synapses in the hippocampus. Moreover, its deletion results in hindrance of newborn neuron migration through a denser ECM network. A novel conditional TIMP2 knockout (KO) model reveals that neuronal TIMP2 regulates adult neurogenesis, accumulation of ECM, and ultimately hippocampus-dependent memory. Our results define a mechanism whereby hippocampus-dependent function is regulated by TIMP2 and its interactions with the ECM to regulate diverse processes associated with synaptic plasticity.

Novel human pluripotent stem cell-derived hypothalamus organoids demonstrate cellular diversity.

The hypothalamus is a region of the brain that plays an important role in regulating body functions and behaviors. There is a growing interest in human pluripotent stem cells (hPSCs) for modeling diseases that affect the hypothalamus. Here, we established an hPSC-derived hypothalamus organoid differentiation protocol to model the cellular diversity of this brain region. Using an hPSC line with a tyrosine hydroxylase (TH)-TdTomato reporter for dopaminergic neurons (DNs) and other TH-expressing cells, we interrogated DN-specific pathways and functions in electrophysiologically active hypothalamus organoids. Single-cell RNA sequencing (scRNA-seq) revealed diverse neuronal and non-neuronal cell types in mature hypothalamus organoids. We identified several molecularly distinct hypothalamic DN subtypes that demonstrated different developmental maturities. Our in vitro 3D hypothalamus differentiation protocol can be used to study the development of this critical brain structure and can be applied to disease modeling to generate novel therapeutic approaches for disorders centered around the hypothalamus.

High-throughput generation of midbrain dopaminergic neuron organoids from reporter human pluripotent stem cells.

Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain. For complete details on the use and execution of this protocol, please refer to Ahfeldt et al. (2020).

Context-dependent expression of a conditionally-inducible form of active Akt.

Akt kinases are key signaling components in proliferation-competent and post-mitotic cells. Here, we sought to create a conditionally-inducible form of active Akt for both in vitro and in vivo applications. We fused a ligand-responsive Destabilizing Domain (DD) derived from E. coli dihydrofolate reductase to a constitutively active mutant form of Akt1, Akt(E40K). Prior work indicated that such fusion proteins may be stabilized and induced by a ligand, the antibiotic Trimethoprim (TMP). We observed dose-dependent, reversible induction of both total and phosphorylated/active DD-Akt(E40K) by TMP across several cellular backgrounds in culture, including neurons. Phosphorylation of FoxO4, an Akt substrate, was significantly elevated after DD-Akt(E40K) induction, indicating the induced protein was functionally active. The induced Akt(E40K) protected cells from apoptosis evoked by serum deprivation and was neuroprotective in two cellular models of Parkinson's disease (6-OHDA and MPP+ exposure). There was no significant protection without induction. We also evaluated Akt(E40K) induction by TMP in mouse substantia nigra and striatum after neuronal delivery via an AAV1 adeno-associated viral vector. While there was significant induction in striatum, there was no apparent induction in substantia nigra. To explore the possible basis for this difference, we examined DD-Akt(E40K) induction in cultured ventral midbrain neurons. Both dopaminergic and non-dopaminergic neurons in the cultures showed DD-Akt(E40K) induction after TMP treatment. However, basal DD-Akt(E40K) expression was 3-fold higher for dopaminergic neurons, resulting in a significantly lower induction by TMP in this population. Such findings suggest that dopaminergic neurons may be relatively inefficient in protein degradation, a property that could relate to their lack of apparent DD-Akt(E40K) induction in vivo and to their selective vulnerability in Parkinson's disease. In summary, we generated an inducible, biologically active form of Akt. The degree of inducibility appears to reflect cellular context that will inform the most appropriate applications for this and related reagents.

A quantitative evaluation of a 2.5-kb rat tyrosine hydroxylase promoter to target expression in ventral mesencephalic dopamine neurons in vivo.

Adeno-associated viruses (AAVs) have become powerful tools in neuroscience for both basic research and potential therapeutic use. They have become especially important tools for optogenetic experiments based on their ability to achieve transgene expression in postmitotic neurons with regional selectivity. With the use of appropriate promoter elements they can achieve cellular specificity as well. One population of neurons that plays a central role in human neurodegenerative and psychiatric diseases are the dopamine neurons of the midbrain. To study these neurons in vivo with advanced techniques it would be highly advantageous to characterize an appropriate specific promoter. To this end we have characterized a 2.5-kb sequence of the rat tyrosine hydroxylase (TH) promoter. The rTHp(2.5) promoter induced expression of the fluorescent reporter protein mCherry in SN dopamine neurons. Although it showed excellent specificity in cortex and striatum, where no reporter expression was observed, in the SN region many neurons expressed reporter but not TH. We show that some of the TH negativity is due to the suppression of its expression by the transgene. We conclude that rTHp(2.5) does preferentially label dopamine neurons but its specificity is not complete within the substantia nigra and caution must be used.

Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo.

The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.

An early axonopathy in a hLRRK2(R1441G) transgenic model of Parkinson disease.

Mutations in the gene for LRRK2 are the most common cause of familial Parkinson's disease (PD) and patients with these mutations manifest clinical features that are indistinguishable from those of the more common sporadic form. Thus, investigations of disease mechanisms based on disease-causing LRRK2 mutations can be expected to shed light on the more common sporadic form as well as the inherited form. We have shown that as human BAC transgenic hLRRK2(R1441G) mice age, they exhibit two abnormalities in the nigrostriatal dopaminergic system: an axonopathy and a diminished number of dendrites in the substantia nigra (SN). To better understand disease mechanisms it is useful to determine where in the affected neural system the pathology first begins. We therefore examined the nigrostriatal dopaminergic system in young mice to determine the initial site of pathology. Brains from hLRRK2(R1441G) and littermate control mice at 2-4months of age were examined by immunohistochemistry, anterograde fluorescent axon labeling and ultrastructural analysis. SN neurons, their projecting axons and the striatal terminal fields were assessed. The first identifiable abnormality in this system is an axonopathy characterized by giant polymorphic axon spheroids, the presence of intra-axonal autophagic vacuoles and intra-axonal myelin invagination. An initial involvement of axons has also been reported for other genetic models of PD. These observations support the concept that axons are involved early in the course of the disease. We suggest that effective neuroprotective approaches will be aimed at preventing axonal degeneration.

Neurotrophic effects of serum- and glucocorticoid-inducible kinase on adult murine mesencephalic dopamine neurons.

Mesencephalic dopamine neurons are central to many aspects of human cognition, motivational, and motor behavior, and they are uniquely vulnerable to degenerative neurologic disorders such as Parkinson's disease. There is growing evidence that in the mature brain these neurons not only remain responsive to neurotrophic support, but are dependent on it for viability and function. Little is known of the cellular signaling pathways that mediate this support, although some evidence suggests that protein kinase Akt/PKB may play such a role. Another candidate for such a role is serum- and glucocorticoid-inducible kinase (SGK), a member of the AGC kinase family that is closely related to Akt. We have herein examined the responsiveness of adult mouse dopamine neurons in vivo to overexpression of wild-type and a constitutively active form of SGK by use of viral vector transfer in normal mice and both before and after 6-OHDA lesion. We find that SGK induces a broad spectrum of neurotrophic effects on these neurons, including induction of neuronal hypertrophy, protection from both neuron death and neurotoxin-induced retrograde axonal degeneration, and axon regeneration. Given the diverse and robust effects of SGK on these neurons, and its abundant expression in them, we suggest that SGK, like closely related Akt, may play a role in their responsiveness to neurotrophic factors and in adult maintenance. It therefore offers a novel target for therapeutic development.

AAV transduction of dopamine neurons with constitutively active Rheb protects from neurodegeneration and mediates axon regrowth.

There are currently no therapies that provide either protection or restoration of neuronal function for adult-onset neurodegenerative diseases such as Parkinson's disease (PD). Many clinical efforts to provide such benefits by infusion of neurotrophic factors have failed, in spite of robust effects in preclinical assessments. One important reason for these failures is the difficulty, due to diffusion limits, of providing these protein molecules in sufficient amounts to the intended cellular targets in the central nervous system. This challenge suggests an alternative approach, that of viral vector transduction to directly activate the intracellular signaling pathways that mediate neurotrophic effects. To this end we have investigated the ability of a constitutively active form of the GTPase Rheb, an important activator of mammalian target of rapamycin (mTor) signaling, to mediate neurotrophic effects in dopamine neurons of the substantia nigra (SN), a population of neurons affected in PD. We find that constitutively active hRheb(S16H) induces many neurotrophic effects in mice, including abilities to both preserve and restore the nigrostriatal dopaminergic axonal projections in a highly destructive neurotoxin model. We conclude that direct viral vector transduction of vulnerable neuronal populations to activate intracellular neurotrophic signaling pathways offers promise for the treatment of neurodegenerative disease.

Age and α-synuclein expression interact to reveal a dependence of dopaminergic axons on endogenous Akt/PKB signaling.

The mechanisms underlying the chronic neurodegeneration that occurs in Parkinson's disease (PD) are unknown. One emerging hypothesis is that neural systems deteriorate and eventually degenerate due to a primary failure of either extrinsic neurotrophic support or the intrinsic cellular pathways that mediate such support. One of the cellular pathways that have been often identified in mediating neurotrophic effects is that of PI3K/Akt signaling. In addition, recent observations have suggested a primary failure of PI3K/Akt signaling in animal models and in PD patients. Therefore, to explore the possible role of endogenous Akt signaling in maintaining the viability and functionality of substantia nigra (SN) dopamine neurons, one of the principal systems affected in PD, we have used an adeno-associated viral vector to transduce them with a dominant negative (DN) form of Akt, the pleckstrin homology (PH) domain alone (DN(PH)-Akt). In addition, we have examined the effect of DN(PH)-Akt in murine models of two risk factors for human PD: advanced age and increased expression of α-synuclein. We find that transduction of these neurons in normal adult mice has no effect on any aspect of their morphology at 4 or 7weeks. However, in both aged mice and in transgenic mice with increased expression of human α-synuclein we observe decreased phenotypic expression of the catecholamine synthetic enzyme tyrosine hydroxylase (TH) in dopaminergic axons and terminals in the striatum. In aged transgenic α-synuclein over-expressing mice this reduction was 2-fold as great. We conclude that the two principal risk factors for human PD, advanced age and increased expression of α-synuclein, reveal a dependence of dopaminergic neurons on endogenous Akt signaling for maintenance of axonal phenotype.

Dopaminergic pathway reconstruction by Akt/Rheb-induced axon regeneration.

OBJECTIVE: A prevailing concept in neuroscience has been that the adult mammalian central nervous system is incapable of restorative axon regeneration. Recent evidence, however, has suggested that reactivation of intrinsic cellular programs regulated by protein kinase B (Akt)/mammalian target of rapamycin (mTor) signaling may restore this ability. METHODS: To assess this possibility in the brain, we have examined the ability of adenoassociated virus (AAV)-mediated transduction of dopaminergic neurons of the substantia nigra (SN) with constitutively active forms of the kinase Akt and the GTPase Ras homolog enriched in brain (Rheb) to induce regrowth of axons after they have been destroyed by neurotoxin lesion. RESULTS: Both constitutively active myristoylated Akt and hRheb(S16H) induce regrowth of axons from dopaminergic neurons to their target, the striatum. Histological analysis demonstrates that these new axons achieve morphologically accurate reinnervation. In addition, functional reintegration into target circuitry is achieved, as indicated by partial behavioral recovery. INTERPRETATION: We conclude that regrowth of axons within the adult nigrostriatal projection, a system that is prominently affected in Parkinson's disease, can be achieved by activation of Akt/mTor signaling in surviving endogenous mesencephalic dopaminergic neurons by viral vector transduction.

Akt suppresses retrograde degeneration of dopaminergic axons by inhibition of macroautophagy.

Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.

Glial cell line-derived neurotrophic factor receptor-α1 expressed in striatum in trans regulates development and injury response of dopamine neurons of the substantia nigra.

Many of the cellular effects of glial cell line-derived neurotrophic factor are initiated by binding to GNDF family receptor alpha-1 (GFRα1), and mediated by diverse intracellular signaling pathways, most notably through the Ret tyrosine kinase. Ret may be activated by the cell autonomous expression of GFRα1 ('in cis'), or by its non-cell autonomous presence ('in trans'), in either a soluble or immobilized state. GFRα1 is expressed in the striatum, a target of the dopaminergic projection of the substantia nigra. To determine whether post-synaptic expression of GFRα1 in striatum in trans has effects on the development or adult responses to injury of dopamine neurons, we have created transgenic mice in which GFRα1 expression is selectively increased in striatum and other forebrain targets of the dopaminergic projection. Post-synaptic GFRα1 has profound effects on the development of dopamine neurons, resulting in a 40% increase in their adult number. This morphologic effect was associated with an augmented motor response to amphetamine. In adult mice, post-synaptic GFRα1 expression did not affect neuron survival following neurotoxic lesion, but it did increase the preservation of striatal dopaminergic innervation. We conclude that post-synaptic striatal GFRα1 expression has important effects on the biology of dopamine neurons in vivo.

Regulation of the postnatal development of dopamine neurons of the substantia nigra in vivo by Akt/protein kinase B.

Following mitosis, specification and migration during embryogenesis, dopamine neurons of the mesencephalon undergo a postnatal naturally occurring cell death event that determines their final adult number, and a period of axonal growth that determines pattern and extent of target contacts. While a number of neurotrophic factors have been suggested to regulate these developmental events, little is known, especially in vivo, of the cell signaling pathways that mediate these effects. We have examined the possible role of Akt/Protein Kinase B by transduction of these neurons in vivo with adeno-associated viral vectors to express either a constitutively active or a dominant negative form of Akt/protein kinase B. We find that Akt regulates multiple features of the postnatal development of these neurons, including the magnitude of the apoptotic developmental cell death event, neuron size, and the extent of target innervation of the striatum. Given the diversity and magnitude of its effects, the regulation of the development of these neurons by Akt may have implications for the many psychiatric and neurologic diseases in which these neurons may play a role.

Antiapoptotic and trophic effects of dominant-negative forms of dual leucine zipper kinase in dopamine neurons of the substantia nigra in vivo.

There is extensive evidence that the mitogen-activated protein kinase (MAPK) signaling cascade mediates programmed cell death in neurons. However, current evidence that the mixed linage kinases (MLKs), upstream in this cascade, mediate cell death is based, in the in vivo context, entirely on pharmacological approaches. The compounds used in these studies have neither complete specificity nor selectivity among these kinases. Therefore, to better address the molecular specificity of the MLKs in mediating neuron death, we used dominant-negative constructs delivered by AAV (adenoassociated virus) vector transfer. We assessed effects in a neurotoxin model of parkinsonism, in which neuroprotection by pharmacologic MLK inhibition has been reported. We find that two dominant-negative forms of dual leucine zipper kinase (DLK) inhibit apoptosis and enhance long-term survival of dopamine neurons, but a dominant negative of MLK3 does not. Interestingly, the kinase-dead form of DLK not only blocks apoptosis but also has trophic effects on dopamine neurons. Although the MAPK cascade activates a number of downstream cell death mediators, we find that inhibition of DLK correlates closely with blockade of phosphorylation of c-jun and prevention of cell death. We conclude that DLK acts primarily through c-jun phosphorylation to mediate cell death in this model.

Oncoprotein Akt/PKB induces trophic effects in murine models of Parkinson's disease.

Despite promising preclinical studies, neurotrophic factors have not yet achieved an established role in the treatment of human neurodegenerative diseases. One impediment has been the difficulty in providing these macromolecules in sufficient quantity and duration at affected sites. An alternative approach is to directly activate, by viral vector transduction, intracellular signaling pathways that mediate neurotrophic effects. We have evaluated this approach in dopamine neurons of the substantia nigra, neurons affected in Parkinson's disease, by adeno-associated virus 1 transduction with a gene encoding a myristoylated, constitutively active form of the oncoprotein Akt/PKB. Adeno-associated virus Myr-Akt has pronounced trophic effects on dopamine neurons of adult and aged mice, including increases in neuron size, phenotypic markers, and sprouting. Transduction confers almost complete protection against apoptotic cell death in a highly destructive neurotoxin model. Activation of intracellular neurotrophic signaling pathways by vector transfer is a feasible approach to neuroprotection and restorative treatment of neurodegenerative disease.