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The family of caspases is known to mediate many cellular pathways beyond cell death, including cell differentiation, axonal pathfinding, and proliferation. Since the identification of the family of cell death proteases, there has been a search for tools to identify and expand the function of specific family members in development, health, and disease states. However, many of the currently commercially available caspase tools that are widely used are not specific for the targeted caspase. In this report, we delineate the approach we have used to identify, validate, and target caspase-9 in the nervous system using a novel inhibitor and genetic approaches with immunohistochemical read-outs. Specifically, we used the retinal neuronal tissue as a model to identify and validate the presence and function of caspases. This approach enables the interrogation of cell-type specific apoptotic and non-apoptotic caspase-9 functions and can be applied to other complex tissues and caspases of interest. Understanding the functions of caspases can help to expand current knowledge in cell biology, and can also be advantageous to identify potential therapeutic targets due to their involvement in disease.

Central nervous system ischemic injury features neuronal dysfunction, inflammation and breakdown of vascular integrity. Here we show that activation of endothelial caspase-9 after hypoxia-ischemia is a critical event in subsequent dysfunction of the blood-retina barrier, using a panel of interrelated ophthalmic in vivo imaging measures in a mouse model of retinal vein occlusion (RVO). Rapid nonapoptotic activation of caspase-9 and its downstream effector caspase-7 in endothelial cells promotes capillary ischemia and retinal neurodegeneration. Topical eye-drop delivery of a highly selective caspase-9 inhibitor provides morphological and functional retinal protection. Inducible endothelial-specific caspase-9 deletion phenocopies this protection, with attenuated retinal edema, reduced inflammation and preserved neuroretinal morphology and function following RVO. These results reveal a non-apoptotic function of endothelial caspase-9 which regulates blood-retina barrier integrity and neuronal survival, and identify caspase-9 as a therapeutic target in neurovascular disease.

Specific therapies for neurologic diseases such as Alzheimer's disease provide the potential for better clinical outcomes. Expression of caspases in the brain is developmentally regulated, and dysregulated in neurologic disease, supporting that caspases may be therapeutic targets. The activity of caspases is carefully regulated via binding partners, cleavage, or endogenous inhibitors to prevent spontaneous activation, which could lead to aberrant cell death. This review serves as a brief examination of the current understanding of the regulation and function of caspases, and approaches to specifically target aberrant caspase activity. The use of proper tools to investigate individual caspases is addressed. Moreover, it summarizes the reports of various caspases in Alzheimer's disease studies. A better understanding of specific caspase pathways in heath and neurodegenerative disease is crucial for identifying specific targets for the development of therapeutic interventions.

Lissencephaly is a malformation of cortical development typically caused by deficient neuronal migration resulting in cortical thickening and reduced gyration. Here we describe a "thin" lissencephaly (TLIS) variant characterized by megalencephaly, frontal predominant pachygyria, intellectual disability, and seizures. Trio-based whole-exome sequencing and targeted re-sequencing identified recessive mutations of CRADD in six individuals with TLIS from four unrelated families of diverse ethnic backgrounds. CRADD (also known as RAIDD) is a death-domain-containing adaptor protein that oligomerizes with PIDD and caspase-2 to initiate apoptosis. TLIS variants cluster in the CRADD death domain, a platform for interaction with other death-domain-containing proteins including PIDD. Although caspase-2 is expressed in the developing mammalian brain, little is known about its role in cortical development. CRADD/caspase-2 signaling is implicated in neurotrophic factor withdrawal- and amyloid-β-induced dendritic spine collapse and neuronal apoptosis, suggesting a role in cortical sculpting and plasticity. TLIS-associated CRADD variants do not disrupt interactions with caspase-2 or PIDD in co-immunoprecipitation assays, but still abolish CRADD's ability to activate caspase-2, resulting in reduced neuronal apoptosis in vitro. Homozygous Cradd knockout mice display megalencephaly and seizures without obvious defects in cortical lamination, supporting a role for CRADD/caspase-2 signaling in mammalian brain development. Megalencephaly and lissencephaly associated with defective programmed cell death from loss of CRADD function in humans implicate reduced apoptosis as an important pathophysiological mechanism of cortical malformation. Our data suggest that CRADD/caspase-2 signaling is critical for normal gyration of the developing human neocortex and for normal cognitive ability.

Caspase 2 was initially identified as a neuronally expressed developmentally down-regulated gene (HUGO gene nomenclature CASP2) and has been shown to be required for neuronal death induced by several stimuli, including NGF (nerve growth factor) deprivation and Aβ (β-amyloid). In non-neuronal cells the PIDDosome, composed of caspase 2 and two death adaptor proteins, PIDD (p53-inducible protein with a death domain) and RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain}, has been proposed as the caspase 2 activation complex, although the absolute requirement for the PIDDosome is not clear. To investigate the requirement for the PIDDosome in caspase-2-dependent neuronal death, we have examined the necessity for each component in induction of active caspase 2 and in execution of caspase-2-dependent neuronal death. We find that both NGF deprivation and Aβ treatment of neurons induce active caspase 2 and that induction of this activity depends on expression of RAIDD, but is independent of PIDD expression. We show that treatment of wild-type or PIDD-null neurons with Aβ or NGF deprivation induces formation of a complex of caspase 2 and RAIDD. We also show that caspase-2-dependent execution of neurons requires RAIDD, not PIDD. Caspase 2 activity can be induced in neurons from PIDD-null mice, and NGF deprivation or Aβ use caspase 2 and RAIDD to execute death of these neurons.

Recent studies demonstrate that the neuropeptide VGF (nonacronymic) is regulated in the hippocampus by antidepressant therapies and animal models of depression and that acute VGF treatment has antidepressant-like activity in animal paradigms. However, the role of VGF in human psychiatric disorders is unknown. We now demonstrate using in situ hybridization that VGF is downregulated in bipolar disorder in the CA region of the hippocampus and Brodmann9s area 9 of the prefrontal cortex. The mechanism of VGF in relation to LiCl was explored. Both LiCl intraperitoneally and VGF intracerebroventricularly reduced latency to drink in novelty-induced hypophagia, and LiCl was not effective in VGF +/− mice, suggesting that VGF may contribute to the effects of LiCl in this behavioral procedure that responds to chronic antidepressant treatment. VGF by intrahippocampal injection also had novel activity in an amphetamine-induced hyperlocomotion assay, thus mimicking the actions of LiCl injected intraperitoneally in a system that phenocopies manic-like behavior. Moreover, VGF +/− mice exhibited increased locomotion after amphetamine treatment and did not respond to LiCl, suggesting that VGF is required for the effects of LiCl in curbing the response to amphetamine. Finally, VGF delivered intracerebroventricularly in vivo activated the same signaling pathways as LiCl and is necessary for the induction of mitogen-activated protein kinase and Akt by LiCl, thus lending insight into the molecular mechanisms underlying the actions of VGF. The dysregulation of VGF in bipolar disorder as well as the behavioral effects of the neuropeptide similar to LiCl suggests that VGF may underlie the pathophysiology of bipolar disorder.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Alzheimer’s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer’s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer’s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

The three-dimensional morphology of the membranous labyrinth of Xenopus laevis is presented from embryonic through late tadpole development (stages 28 to 52, inclusive). This was accomplished by paint-filling the endolymphatic spaces of Xenopus ears at a series of stages, beginning with the embryonic otic vesicle and ending with the complex ear of the late tadpole. At stage 52, the inner ear has expanded approximately 23-fold in its anterior/posterior dimension compared with stage 28 and it is a miniature of the adult form. The paint-filling technique illustrates the dramatic changes required to convert a simple ear vesicle into the elaborate form of the adult, including semicircular canal formation and genesis of vestibular and auditory organs, and it can serve as a basis for phenotype identification in experimentally or genetically manipulated ears.

SummaryIn Alzheimer’s disease (AD) brain, exposure of axons to Aβ causes pathogenic changes that spread retrogradely by unknown mechanisms, affecting the entire neuron. We found that locally applied Aβ1-42 initiates axonal synthesis of a defined set of proteins including the transcription factor ATF4. Inhibition of local translation and retrograde transport or knockdown of axonal Atf4 mRNA abolished Aβ-induced ATF4 transcriptional activity and cell loss. Aβ1-42 injection into the dentate gyrus (DG) of mice caused loss of forebrain neurons whose axons project to the DG. Protein synthesis and Atf4 mRNA were upregulated in these axons, and coinjection of Atf4 siRNA into the DG reduced the effects of Aβ1-42 in the forebrain. ATF4 protein and transcripts were found with greater frequency in axons in the brain of AD patients. These results reveal an active role for intra-axonal translation in neurodegeneration and identify ATF4 as a mediator for the spread of AD pathology.

Caspase-2 is essential for c-Jun transcriptional activation and Bim induction in neuron death. Jean YY1, Ribe EM, Pero ME, Moskalenko M, Iqbal Z, Marks LJ, Greene LA, Troy CM. Neuronal apoptotic death generally requires de novo transcription, and activation of the transcription factor c-Jun has been shown to be necessary in multiple neuronal death paradigms. Caspase-2 has been implicated in death of neuronal and non-neuronal cells, but its relationship to transcriptional activation has not been clearly elucidated. In the present study, using two different neuronal apoptotic paradigms, β-amyloid treatment and NGF (nerve growth factor) withdrawal, we examined the hierarchical role of caspase-2 activation in the transcriptional control of neuron death. Both paradigms induce rapid activation of caspase-2 as well as activation of the transcription factor c-Jun and subsequent induction of the pro-apoptotic BH3 (Bcl-homology domain 3)-only protein Bim (Bcl-2-interacting mediator of cell death). Caspase-2 activation is dependent on the adaptor protein RAIDD {RIP (receptor-interacting protein)-associated ICH-1 [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with a death domain}, and both caspase-2 and RAIDD are required for c-Jun activation and Bim induction. The present study thus shows that rapid caspase-2 activation is essential for c-Jun activation and Bim induction in neurons subjected to apoptotic stimuli. This places caspase-2 at an apical position in the apoptotic cascade and demonstrates for the first time that caspase-2 can regulate transcription.

Caspases, initially identified as a family of proteases regulating cell death, have been found to have nonapoptotic functions as well. Some family members are critical for mediating programmed cell death in development. After development, caspases are downregulated in the nervous system, but continue to perform important nonapoptotic functions relevant for neurogenesis and synaptic plasticity. In neurodegenerative diseases, where aberrant neuronal death is an outstanding feature, there is an increase in caspase activity. The specific caspase death pathways leading to dysfunction and death have still not been fully clarified, despite the plethora of scientific literature addressing these issues. In this chapter, we will present the current knowledge of caspase activation and activity pathways, the current tools for examining caspases, and functions of caspases in the nervous system in health and in disease. Alzheimer's Disease, the most common neurodegenerative disorder, and cerebral ischemia, the most common cause of neurologic death, are used to illustrate our current understanding of death signaling in neurodegenerative diseases. A better understanding of how caspases function in health and disease would provide appropriate specific targets for the development of therapeutic interventions for these diseases. Life and death are exquisitely regulated at the cellular level from development through maturity. During development, neuronal death is the major factor shaping the nervous system. This death is mainly caspase-mediated apoptosis. Once the waves of developmental death have passed (death occurs at different times in different parts of the nervous system), there is downregulation of the death machinery, as the postmitotic neurons should live for the life of the organism. Aberrant neuronal death is a major part of neurodegenerative disorders, but there is still no clear understanding of the processes leading to the phenotypes of the various diseases. Even the type of death that occurs continues to be debated, whether it is apoptotic, necrotic, or autophagic, or some combination of these death mechanisms. Here, we will discuss the role that the caspases play in neuronal function, dysfunction, and death. First, we will discuss the regulation of caspase activation and activity. We will examine the current understanding of caspase function in developmental neuronal death and then illustrate the role of caspases in neuronal death in disease employing two diseases of neuronal loss, Alzheimer's Disease (AD), which is the most common chronic neurodegenerative disorder, and cerebral ischemia/stroke, the third most common cause of death in Western society, which is an acute neuronal disorder with chronic sequelae.

Caspases, initially identified as a family of proteases regulating cell death, have been found to have nonapoptotic functions as well. Some family members are critical for mediating programmed cell death in development. After development, caspases are downregulated in the nervous system, but continue to perform important nonapoptotic functions relevant for neurogenesis and synaptic plasticity. In neurodegenerative diseases, where aberrant neuronal death is an outstanding feature, there is an increase in caspase activity. The specific caspase death pathways leading to dysfunction and death have still not been fully clarified, despite the plethora of scientific literature addressing these issues. In this chapter, we will present the current knowledge of caspase activation and activity pathways, the current tools for examining caspases, and functions of caspases in the nervous system in health and in disease. Alzheimer's Disease, the most common neurodegenerative disorder, and cerebral ischemia, the most common cause of neurologic death, are used to illustrate our current understanding of death signaling in neurodegenerative diseases. A better understanding of how caspases function in health and disease would provide appropriate specific targets for the development of therapeutic interventions for these diseases. Life and death are exquisitely regulated at the cellular level from development through maturity. During development, neuronal death is the major factor shaping the nervous system. This death is mainly caspase-mediated apoptosis. Once the waves of developmental death have passed (death occurs at different times in different parts of the nervous system), there is downregulation of the death machinery, as the postmitotic neurons should live for the life of the organism. Aberrant neuronal death is a major part of neurodegenerative disorders, but there is still no clear understanding of the processes leading to the phenotypes of the various diseases. Even the type of death that occurs continues to be debated, whether it is apoptotic, necrotic, or autophagic, or some combination of these death mechanisms. Here, we will discuss the role that the caspases play in neuronal function, dysfunction, and death. First, we will discuss the regulation of caspase activation and activity. We will examine the current understanding of caspase function in developmental neuronal death and then illustrate the role of caspases in neuronal death in disease employing two diseases of neuronal loss, Alzheimer's Disease (AD), which is the most common chronic neurodegenerative disorder, and cerebral ischemia/stroke, the third most common cause of death in Western society, which is an acute neuronal disorder with chronic sequelae.

A key neurotrophin responsible for the survival and function of basal forebrain (BF) cholinergic neurons is brain-derived neurotrophic factor (BDNF). A number of studies now indicate that a source of this factor may be BF astrocytes. This study was designed to define the role of BF-astrocyte-derived BDNF on cholinergic neurons. Moreover, it investigated regulatory events that modulate BDNF content and release. In initial work BDNF derived from BF-astrocyte-conditioned medium (ACM) was found to increase both numbers of BF acetylcholinesterase (AChE+) cholinergic neurons and the cholinergic synthetic enzyme choline acetyltransferase (ChAT). Western blots, immunocytochemistry and pharmacological inhibition studies revealed that glutamate, through group I metabotropic glutamate receptors (mGluR), increases the intracellular levels of BDNF in BF astrocytes in culture, as well as its release. Furthermore, the release of BDNF is mediated by the actions of PLC, IP3 and internal stores of Ca2+. These results suggest that BF astrocytes serve as local sources of BDNF for cholinergic neurons, and that they may be regulated as such by the neuronal signal, glutamate, through the mediation of group I metabotropic receptors and the PLC pathway.

It is now quite clear that as part of their support role, astrocytes produce and release multiple proteins that impact survival, migration, differentiation, and function of proximate neurons. This astrocyte role is particularly evident during development, remains in play in the normal adult brain, and is enhanced after injury. The molecules produced include those known traditionally as cytokines, chemokines, and trophic factors. Importantly, these proteins can serve as trophic or toxic agents and may be differentially regulated by the signals impacting on astrocytes at various stages of brain development and maturity (Fig. 13.1 ). The story of proteins produced by astrocytes, then, is a complex one. Multiple trophic or toxic molecules