Genetically Encoded and Modular SubCellular Organelle Probes (GEM-SCOPe) reveal lysosomal and mitochondrial dysfunction driven by knockout.

Cellular processes including lysosomal and mitochondrial dysfunction are implicated in the development of many diseases. Quantitative visualization of mitochondria and lysosoesl is crucial to understand how these organelles are dysregulated during disease. To address a gap in live-imaging tools, we developed GEM-SCOPe (Genetically Encoded and Modular SubCellular Organelle Probes), a modular toolbox of fluorescent markers designed to inform on localization, distribution, turnover, and oxidative stress of specific organelles.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.