Linda Hassinger

Abnormalities in Mitochondrial Structure in Cells from Patients with Bipolar Disorder

Postmortem, genetic, brain imaging, and peripheral cell studies all support decreased mitochondrial activity as a factor in the manifestation of Bipolar Disorder (BD). Because abnormal mitochondrial morphology is often linked to altered energy metabolism, we investigated whether changes in mitochondrial structure were present in brain and peripheral cells of patients with BD. Mitochondria from patients with BD exhibited size and distributional abnormalities compared with psychiatrically-healthy age-matched controls. Specifically, in brain, individual mitochondria profiles had significantly smaller areas, on average, in BD samples (P = 0.03). In peripheral cells, mitochondria in BD samples were concentrated proportionately more within the perinuclear region than in distal processes (P = 0.0008). These mitochondrial changes did not appear to be correlated with exposure to lithium. Also, these abnormalities in brain and peripheral cells were independent of substantial changes in the actin or tubulin cytoskeleton with which mitochondria interact. The observed changes in mitochondrial size and distribution may be linked to energy deficits and, therefore, may have consequences for cell plasticity, resilience, and survival in patients with BD, especially in brain, which has a high-energy requirement. The findings may have implications for diagnosis, if they are specific to BD, and for treatment, if they provide clues as to the underlying pathophysiology of BD.

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L–null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H–null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons.

Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area

Significance Hypocretin (orexin) and dynorphin are neuromodulators that play an important role in regulating affect and motivation. Orexin is critical for reward and is implicated in drug seeking, whereas dynorphin mediates negative mood and is implicated in depressive-like states. Considering these opposing effects, reports that both peptides are expressed in the same neurons and are coreleased are counterintuitive. Here, we demonstrate that orexin and dynorphin are coexpressed within the same synaptic vesicles and that this colocalization has a profound influence on reward, drug taking, and impulsive-like behavior. The fact that orexin occludes the depressive-like antireward effects of dynorphin significantly changes how we view the functional role of orexin in the brain. Hypocretin (orexin) and dynorphin are neuropeptides with opposing actions on motivated behavior. Orexin is implicated in states of arousal and reward, whereas dynorphin is implicated in depressive-like states. We show that, despite their opposing actions, these peptides are packaged in the same synaptic vesicles within the hypothalamus. Disruption of orexin function blunts the rewarding effects of lateral hypothalamic (LH) stimulation, eliminates cocaine-induced impulsivity, and reduces cocaine self-administration. Concomitant disruption of dynorphin function reverses these behavioral changes. We also show that orexin and dynorphin have opposing actions on excitability of ventral tegmental area (VTA) dopamine neurons, a prominent target of orexin-containing neurons, and that intra-VTA orexin antagonism causes decreases in cocaine self-administration and LH self-stimulation that are reversed by dynorphin antagonism. Our findings identify a unique cellular process by which orexin can occlude the reward threshold-elevating effects of coreleased dynorphin and thereby act in a permissive fashion to facilitate reward.

Down Syndrome Fibroblast Model of Alzheimer-Related Endosome Pathology: Accelerated Endocytosis Promotes Late Endocytic Defects

Endocytic dysfunction is an early pathological change in Alzheimers disease (AD) and Downs syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains.

APP-BP1 mediates APP-induced apoptosis and DNA synthesis and is increased in Alzheimer's disease brain.

APP-BP1, first identified as an amyloid precursor protein (APP) binding protein, is the regulatory subunit of the activating enzyme for the small ubiquitin-like protein NEDD8. We have shown that APP-BP1 drives the S- to M-phase transition in dividing cells, and causes apoptosis in neurons (Chen, Y., D.L. McPhie, J. Hirschberg, and R.L. Neve. 2000. J. Biol. Chem. 275:8929–8935). We now demonstrate that APP-BP1 binds to the COOH-terminal 31 amino acids of APP (C31) and colocalizes with APP in a lipid-enriched fraction called lipid rafts. We show that coexpression of a peptide representing the domain of APP-BP1 that binds to APP, abolishes the ability of overexpressed APP or the V642I mutant of APP to cause neuronal apoptosis and DNA synthesis. A dominant negative mutant of the NEDD8 conjugating enzyme hUbc12, which participates in the ubiquitin-like pathway initiated by APP-BP1, blocks neuronal apoptosis caused by APP, APP(V642I), C31, or overexpression of APP-BP1. Neurons overexpressing APP or APP(V642I) show increased APP-BP1 protein levels in lipid rafts. A similar increase in APP-BP1 in lipid rafts is observed in the Alzheimers disease brain hippocampus, but not in less-affected areas of Alzheimers disease brain. This translocation of APP-BP1 to lipid rafts is accompanied by a change in the subcellular localization of the ubiquitin-like protein NEDD8, which is activated by APP-BP1.

Subpopulations of neurons expressing parvalbumin in the human amygdala.

Amygdalar intrinsic inhibitory networks comprise several subpopulations of γ‐aminobutyric acidergic neurons, each characterized by distinct morphological features and clusters of functionally relevant neurochemical markers. In rodents, the calcium‐binding proteins parvalbumin (PVB) and calbindin D28k (CB) are coexpressed in large subpopulations of amygdalar interneurons. PVB‐immunoreactive (‐IR) neurons have also been shown to be ensheathed by perineuronal nets (PNN), extracellular matrix envelopes believed to affect ionic homeostasis and synaptic plasticity. We tested the hypothesis that differential expression of these three markers may define distinct neuronal subpopulations within the human amygdala. Toward this end, triple‐fluorescent labeling using antisera raised against PVB and CB as well as biotinylated Wisteria floribunda lectin for detection of PNN was combined with confocal microscopy. Among the 1,779 PVB‐IR neurons counted, 18% also expressed CB, 31% were ensheathed in PNN, and 7% expressed both CB and PNN. Forty‐four percent of PVB‐IR neurons did not colocalize with either CB or PNN. The distribution of each of these neuronal subgroups showed substantial rostrocaudal gradients. Furthermore, distinct morphological features were found to characterize each neuronal subgroup. In particular, significant differences relative to the distribution and morphology were detected between PVB‐IR neurons expressing CB and PVB‐IR neurons wrapped in PNNs. These results indicate that amygdalar PVB‐IR neurons can be subdivided into at least four different subgroups, each characterized by a specific neurochemical profile, morphological characteristics, and three‐dimensional distribution. Such properties suggest that each of these neuronal subpopulations may play a specific role within the intrinsic circuitry of the amygdala. J. Comp. Neurol. 496:706–722, 2006.

Detection of Intranasally Delivered Bone Marrow-Derived Mesenchymal Stromal Cells in the Lesioned Mouse Brain: A Cautionary Report

Bone marrow-derived mesenchymal stromal cells (MSCs) hold promise for autologous treatment of neuropathologies. Intranasal delivery is relatively noninvasive and has recently been reported to result in transport of MSCs to the brain. However, the ability of MSCs to migrate from nasal passages to sites of neuropathology and ultimately survive has not been fully examined. In this paper, we harvested MSCs from transgenic mice expressing enhanced green fluorescent protein (cells hereafter referred to as MSC-EGFP) and delivered them intranasally to wild-type mice sustaining mechanical lesions in the striatum. Using fluorescent, colorimetric, and ultrastructural detection methods, GFP-expressing cells were undetectable in the brain from 3 hours to 2 months after MSC delivery. However, bright autofluorescence that strongly resembled emission from GFP was observed in the olfactory bulb and striatum of lesioned control and MSC-EGFP-treated mice. In a control experiment, we directly implanted MSC-EGFPs into the mouse striatum and detected robust GFP expression 1 and 7 days after implantation. These findings suggest that—under our conditions—intranasally delivered MSC-EGFPs do not survive or migrate in the brain. Furthermore, our observations highlight the necessity of including appropriate controls when working with GFP as a cellular marker.

Dissociation of Axonal Neurofilament Content from Its Transport Rate

The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with previous evidence suggesting that the rate of incorporation of transported NF precursors into a metabolically stable stationary cytoskeletal network is the major determinant of axonal NF content, enabling the generation of the striking local variations in NF number seen along axons.

P4-098 : In vivo turnover of retrogradely transported proteins by autophagy is impaired in PS/APP transgenic mice

Introduction: Autophagy, a major pathway for protein and organelle turnover, is induced in neurons as a critical survival response in Alzheimer’s disease (AD). Autophagic vacuoles (AVs), which are capable of generating amyloid-beta, peptide (Yu et al., JCB, 2005), massively accumulate within dystrophic neurites. The significance of these AV accumulations for neurite regeneration or degeneration and their capacity for protein degradation are still unclear. Objective: We investigated in PS/ APP transgenic mice in vivo whether AVs in dystrophic neurites can still interact with endocytic and lysosomal compartments undergoing transport and efficiently degrade proteins. Methods/Results: We used endocytic uptake of native horseradish peroxidase (HRP) after brief intraventricular infusion in wild-type and PS/APP mice to track the dendritic transport and fate of endocytic vesicles in vivo over time (5-240 min). In sections of perfusion-fixed brains analyzed by EM, HRP was detected mainly extracellularly at 5 minutes post-infusion but by 1 hour, was principally intracellular, appearing along dendrites within numerous small ( 100nm) endocytic vesicles. In PS/APP mice, HRP also appeared by 1 hour in the large (0.5-2.0 microns) AVs accumulated within dystrophic neurites as well as in the extracellular space specifically around these neurites. By 4 hours, HRP had largely disappeared from small endocytic vesicles in dendrites of wild-type mice and was principally in rare lipofuscin granules in perikarya, suggesting relatively rapid turnover. By contrast, HRP was still detected within many AVs in dystrophic neurites of PS/APP mice, although HRP distribution within AVs and changes in the maturation states of HRP -containing AVs suggested a continued, albeit slowed, autophagic degradative process. Conclusions: Vesicular trafficking and fusion events within dystrophic neurites remain remarkably dynamic. Endocytic vesicles translocate along dendrites and either fuse with, or are sequestered by, autophagosomes accumulated within the dystrophic neurite. Compared to the highly efficient autophagy in normal neurons, however, autophagic protein degradation in dystrophic neurites of PS/APP mice is inefficient. The highly active transport of APP-rich organelles, continued fusion with AVs in dystrophic neurites, and delays in the complete lysosomal elimination by autophagic substrates provide highly favorable conditions for amyloidbeta, peptide production at these sites.