Mona Buhusi

CHL1 promotes Sema3A‐induced growth cone collapse and neurite elaboration through a motif required for recruitment of ERM proteins to the plasma membrane

Close homolog of L1 (CHL1) is a transmembrane cell adhesion molecule with unique developmental functions in cortical neuronal positioning and dendritic projection within the L1 family, as well as shared functions in promotion of integrin‐dependent neurite outgrowth and semaphorin3A (Sema3A)‐mediated axon repulsion. The molecular mechanisms by which CHL1 mediates these diverse functions are obscure. Here it is demonstrated using a cytofluorescence assay that CHL1 is able to recruit ezrin, a member of the ezrin‐radixin‐moesin (ERM) family of filamentous actin binding proteins to the plasma membrane, and that this requires a membrane‐proximal motif (RGGKYSV) in the CHL1 cytoplasmic domain. This sequence in CHL1 is shown to have novel functions necessary for Sema3A‐induced growth cone collapse and CHL1‐dependent neurite outgrowth and branching in cortical embryonic neurons. In addition, stimulation of haptotactic cell migration and cellular adhesion to fibronectin by CHL1 depends on the CHL1/ERM recruitment motif. These findings suggest that a direct or indirect interaction between CHL1 and ERM proteins mediates Sema3A‐induced growth cone collapse as well as neurite outgrowth and branching, which are essential determinants of axon guidance and connectivity in cortical development.

L1 Interaction with Ankyrin Regulates Mediolateral Topography in the Retinocollicular Projection

Dynamic modulation of adhesion provided by anchorage of axonal receptors with the cytoskeleton contributes to attractant or repellent responses that guide axons to topographic targets in the brain. The neural cell adhesion molecule L1 engages the spectrin-actin cytoskeleton through reversible linkage of its cytoplasmic domain to ankyrin. To investigate a role for L1 association with the cytoskeleton in topographic guidance of retinal axons to the superior colliculus, a novel mouse strain was generated by genetic knock-in that expresses an L1 point mutation (Tyr1229His) abolishing ankyrin binding. Axon tracing revealed a striking mistargeting of mutant ganglion cell axons from the ventral retina, which express high levels of ephrinB receptors, to abnormally lateral sites in the contralateral superior colliculus, where they formed multiple ectopic arborizations. These axons were compromised in extending interstitial branches in the medial direction, a normal response to the high medial to low lateral SC gradient of ephrinB1. Furthermore, ventral but not dorsal L1(Y1229H) retinal cells were impaired for ephrinB1-stimulated adhesion through β1 integrins in culture. The retinocollicular phenotype of the L1(Tyr1229His) mutant provides the first evidence that L1 regulates topographic mapping of retinal axons through adhesion mediated by linkage to the actin cytoskeleton and functional interaction with the ephrinB/EphB targeting system.

Interval Timing Accuracy and Scalar Timing in C57BL/6 Mice

In many species, interval timing behavior is accurate-appropriate estimated durations-and scalar-errors vary linearly with estimated durations. Whereas accuracy has been previously examined, scalar timing has not been clearly demonstrated in house mice (Mus musculus), raising concerns about mouse models of human disease. The authors estimated timing accuracy and precision in C57BL/6 mice, the most used background strain for genetic models of human disease, in a peak-interval procedure with multiple intervals. Both when timing 2 intervals (Experiment 1) or 3 intervals (Experiment 2), C57BL/6 mice demonstrated varying degrees of timing accuracy. An important finding was that, both at the individual and group levels, their precision varied linearly with the subjective estimated duration. Further evidence for scalar timing was obtained using an intraclass correlation statistic. This is the first report of consistent, reliable scalar timing in a sizable sample of house mice, thus validating the peak-interval procedure as a valuable technique, the intraclass correlation statistic as a powerful test of the scalar property, and the C57BL/6 strain as a suitable background for behavioral investigations of genetically engineered mice modeling disorders of interval timing.

ALCAM Regulates Mediolateral Retinotopic Mapping in the Superior Colliculus

ALCAM [activated leukocyte cell adhesion molecule (BEN/SC-1/DM-GRASP)] is a transmembrane recognition molecule of the Ig superfamily (IgSF) containing five Ig domains (two V-type, three C2-type). Although broadly expressed in the nervous and immune systems, few of its developmental functions have been elucidated. Because ALCAM has been suggested to interact with the IgSF adhesion molecule L1, a determinant of retinocollicular mapping, we hypothesized that ALCAM might direct topographic targeting to the superior colliculus (SC) by serving as a substrate within the SC for L1 on incoming retinal ganglion cell (RGC) axons. ALCAM was expressed in the SC during RGC axon targeting and on RGC axons as they formed the optic nerve; however, it was downregulated distally on RGC axons as they entered the SC. Axon tracing with DiI revealed pronounced mistargeting of RGC axons from the temporal retina half of ALCAM null mice to abnormally lateral sites in the contralateral SC, in which these axons formed multiple ectopic termination zones. ALCAM null mutant axons were specifically compromised in medial orientation of interstitial branches, which is known to require the ankyrin binding function of L1. As a substrate, ALCAM–Fc protein promoted L1-dependent attachment of acutely dissociated retinal cells and an L1-expressing, ALCAM-negative cell line, consistent with an ALCAM–L1 heterophilic molecular interaction. Together, these results suggest a model in which ALCAM in the SC interacts with L1 on RGC axons to promote medial extension of RGC axon branches important for mediolateral axon targeting in the formation of retinocollicular maps.

Nerve growth factor metabolic dysfunction in Down’s syndrome brains

Basal forebrain cholinergic neurons play a key role in cognition. This neuronal system is highly dependent on NGF for its synaptic integrity and the phenotypic maintenance of its cell bodies. Basal forebrain cholinergic neurons progressively degenerate in Alzheimers disease and Downs syndrome, and their atrophy contributes to the manifestation of dementia. Paradoxically, in Alzheimers disease brains, the synthesis of NGF is not affected and there is abundance of the NGF precursor, proNGF. We have shown that this phenomenon is the result of a deficit in NGFs extracellular metabolism that compromises proNGF maturation and exacerbates its subsequent degradation. We hypothesized that a similar imbalance should be present in Downs syndrome. Using a combination of quantitative reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting and zymography, we investigated signs of NGF metabolic dysfunction in post-mortem brains from the temporal (n = 14), frontal (n = 34) and parietal (n = 20) cortex obtained from subjects with Downs syndrome and age-matched controls (age range 31-68 years). We further examined primary cultures of human foetal Downs syndrome cortex (17-21 gestational age weeks) and brains from Ts65Dn mice (12-22 months), a widely used animal model of Downs syndrome. We report a significant increase in proNGF levels in human and mouse Downs syndrome brains, with a concomitant reduction in the levels of plasminogen and tissue plasminogen activator messenger RNA as well as an increment in neuroserpin expression; enzymes that partake in proNGF maturation. Human Downs syndrome brains also exhibited elevated zymogenic activity of MMP9, the major NGF-degrading protease. Our results indicate a failure in NGF precursor maturation in Downs syndrome brains and a likely enhanced proteolytic degradation of NGF, changes which can compromise the trophic support of basal forebrain cholinergic neurons. The alterations in proNGF and MMP9 were also present in cultures of Downs syndrome foetal cortex; suggesting that this trophic compromise may be amenable to rescue, before frank dementia onset. Our study thus provides a novel paradigm for cholinergic neuroprotection in Alzheimers disease and Downs syndrome.

Cholinergic Degeneration and Alterations in the TrkA and p75NTR Balance as a Result of Pro-NGF Injection into Aged Rats

Learning and memory impairments occurring with Alzheimers disease (AD) are associated with degeneration of the basal forebrain cholinergic neurons (BFCNs). BFCNs extend their axons to the hippocampus where they bind nerve growth factor (NGF) which is retrogradely transported to the cell body. While NGF is necessary for BFCN survival and function via binding to the high-affinity receptor TrkA, its uncleaved precursor, pro-NGF has been proposed to induce neurodegeneration via binding to the p75NTR and its coreceptor sortilin. Basal forebrain TrkA and NGF are downregulated with aging while pro-NGF is increased. Given these data, the focus of this paper was to determine a mechanism for how pro-NGF accumulation may induce BFCN degeneration. Twenty-four hours after a single injection of pro-NGF into hippocampus, we found increased hippocampal p75NTR levels, decreased hippocampal TrkA levels, and cholinergic degeneration. The data suggest that the increase in p75NTR with AD may be mediated by elevated pro-NGF levels as a result of decreased cleavage, and that pro-NGF may be partially responsible for age-related degenerative changes observed in the basal forebrain. This paper is the first in vivo evidence that pro-NGF can affect BFCNs and may do so by regulating expression of p75NTR neurotrophin receptors.

RPE65 Is Present in Human Green/Red Cones and Promotes Photopigment Regeneration in an In Vitro Cone Cell Model

RPE65 is an abundantly expressed protein within the retinal pigment epithelium (RPE) of the eye that is required for retinoid metabolism to support vision. Its genetic mutations are linked to the congenital disease Leber congenital amaurosis Type 2 (LCA2) characterized by the early onset of central vision loss. Current gene therapy trials have targeted restoration of functional RPE65 within the RPE of these patients with some success. Recent data show that RPE65 is also present within mouse cones to promote function. In this study, we evaluated the presence of RPE65 in human cones and investigated its potential mechanism for supporting cone function in the 661W cone cell line. We found that RPE65 was selectively expressed in human green/red cones but absent from blue cones and mediated ester hydrolysis for photopigment synthesis in vitro. These data suggest that cone RPE65 supports human diurnal vision, potentially enhancing our strategies for treating LCA2.

The nigrostriatal dopamine system of aging GFRα-1 heterozygous mice: neurochemistry, morphology and behavior

Given the established importance of glial cell line‐derived neurotrophic factor (GDNF) in maintaining dopaminergic neurotransmitter systems, the nigrostriatal system and associated behaviors of mice with genetic reduction of its high‐affinity receptor, GDNF receptor (GFR)α‐1 (GFRα‐1+/−), were compared with wild‐type controls. Motor activity and the stimulatory effects of a dopamine (DA) D1 receptor agonist (SKF 82958) were assessed longitudinally at 8 and 18 months of age. Monoamine concentrations and dopaminergic nerve terminals in the striatum and the number of dopaminergic neurons in the substantia nigra (SN) were assessed. The results support the importance of GFRα‐1 in maintaining normal function of the nigrostriatal dopaminergic system, with deficits being observed for GFRα‐1+/− mice at both ages. Motor activity was lower and the stimulatory effects of the DA agonist were enhanced for the older GFRα‐1+/− mice. DA in the striatum was reduced in the GFRα‐1+/− mice at both ages, and tyrosine hydroxylase‐positive cell numbers in the SN were reduced most substantially in the older GFRα‐1+/− mice. The combined behavioral, pharmacological probe, neurochemical and morphological measures provide evidence of abnormalities in GFRα‐1+/− mice that are indicative of an exacerbated aging‐related decline in dopaminergic system function. The noted deficiencies, in turn, suggest that GFRα‐1 is necessary for GDNF to maintain normal function of the nigrostriatal dopaminergic system. Although the precise mechanism(s) for the aging‐related changes in the dopaminergic system remain to be established, the present study clearly establishes that genetic reductions in GFRα‐1 can contribute to the degenerative changes observed in this system during the aging process.

Dissociation of the role of the prelimbic cortex in interval timing and resource allocation: beneficial effect of norepinephrine and dopamine reuptake inhibitor nomifensine on anxiety-inducing distraction

Emotional distracters impair cognitive function. Emotional processing is dysregulated in affective disorders such as depression, phobias, schizophrenia, and post-traumatic stress disorder (PTSD). Among the processes impaired by emotional distracters, and whose dysregulation is documented in affective disorders, is the ability to time in the seconds-to-minutes range, i.e., interval timing. Presentation of task-irrelevant distracters during a timing task results in a delay in responding suggesting a failure to maintain subjective time in working memory, possibly due to attentional and working memory resources being diverted away from timing, as proposed by the Relative Time-Sharing (RTS) model. We investigated the role of the prelimbic cortex in the detrimental effect of anxiety-inducing task-irrelevant distracters on the cognitive ability to keep track of time, using local infusions of norepinephrine and dopamine reuptake inhibitor (NDRI) nomifensine in a modified peak-interval procedure with neutral and anxiety-inducing distracters. Given that some anti-depressants have beneficial effects on attention and working memory, e.g., decreasing emotional response to negative events, we hypothesized that nomifensine would improve maintenance of information in working memory in trials with distracters, resulting in a decrease of the disruptive effect of emotional events on the timekeeping abilities. Our results revealed a dissociation of the effects of nomifensine infusion in prelimbic cortex between interval timing and resource allocation, and between neutral and anxiety-inducing distraction. Nomifensine was effective only during trials with distracters, but not during trials without distracters. Nomifensine reduced the detrimental effect of the distracters only when the distracters were anxiety-inducing, but not when they were neutral. Results are discussed in relation to the brain circuits involved in RTS of resources, and the pharmacological management of affective disorders.

Impaired Interval Timing and Spatial–Temporal Integration in Mice Deficient in CHL1, a Gene Associated with Schizophrenia

Interval timing is crucial for decision-making and motor control and is impaired in many neuropsychiatric disorders, including schizophrenia - a neurodevelopmental disorder with a strong genetic component. Several gene mutations, polymorphisms or rare copy number variants have been associated with schizophrenia. L1 cell adhesion molecules (L1CAMs) are involved in neurodevelopmental processes, and in synaptic function and plasticity in the adult brain. Mice deficient in the Close Homolog to L1 (CHL1) adhesion molecule show alterations of hippocampal and thalamo-cortical neuroanatomy as well as deficits in sensorimotor gating and exploratory behavior. We analyzed interval timing and attentional control of temporal and spatial information in male CHL1 deficient (KO) mice and wild type (WT) controls. In a 20-s peak-interval timing procedure (standard and reversed), KO mice showed a maintained leftward shift of the response function relative to WT, indicative of a deficit in memory encoding/decoding. In trials with 2, 5, or 10-s gaps, KO mice shifted their peak times less than WT controls at longer gap durations, suggesting a decreased (attentional) effect of interruptions. In the spatial-temporal task, KO mice made more working and reference memory errors than controls, suggestive of impaired use of spatial and/or temporal information. When the duration spent on the central platform of the maze was manipulated, WT mice showed fewer spatial errors at the trained duration than at shorter or longer durations, indicative of discrimination based upon spatial-temporal integration. In contrast, performance was similar at all tested durations in KO mice, indicative of control by spatial cues, but not by temporal cues. These results suggest that CHL1 KO mice selectively attend to the more relevant cues of the task, and fail to integrate more complex spatial-temporal information, possibly as a result of reduced memory capacity related to hippocampal impairment, and altered temporal-integration mechanisms possibly due to thalamo-cortical anomalies.