A Photoactivated Protein Degrader for Optical Control of Synaptic Function.

Hundreds of proteins determine the function of synapses, and synapses define the neuronal circuits that subserve myriad brain, cognitive, and behavioral functions. It is thus necessary to precisely manipulate specific proteins at specific sub-cellular locations and times to elucidate the roles of particular proteins and synapses in brain function. We developed PHOtochemically TArgeting Chimeras (PHOTACs) as a strategy to optically degrade specific proteins with high spatial and temporal precision. PHOTACs are small molecules that, upon wavelength-selective illumination, catalyze ubiquitylation and degradation of target proteins through endogenous proteasomes. Here we describe the design and chemical properties of a PHOTAC that targets Ca 2+ /calmodulin-dependent protein kinase II alpha (CaMKIIα), which is abundant and crucial for baseline synaptic function of excitatory neurons. We validate the PHOTAC strategy, showing that the CaMKIIα-PHOTAC is effective in mouse brain tissue. Light activation of CaMKIIα-PHOTAC removed CaMKIIα from regions of the mouse hippocampus only within 25 µm of the illuminated brain surface. The optically-controlled degradation decreases synaptic function within minutes of light activation, measured by the light-initiated attenuation of evoked field excitatory postsynaptic potential (fEPSP) responses to physiological stimulation. The PHOTACs methodology should be broadly applicable to other key proteins implicated in synaptic function, especially for evaluating their precise roles in the maintenance of long-term potentiation and memory within subcellular dendritic domains.

Restoring Light Sensitivity in Blind Retinae Using a Photochromic AMPA Receptor Agonist.

Retinal degenerative diseases can have many possible causes and are currently difficult to treat. As an alternative to therapies that require genetic manipulation or the implantation of electronic devices, photopharmacology has emerged as a viable approach to restore visual responses. Here, we present a new photopharmacological strategy that relies on a photoswitchable excitatory amino acid, ATA. This freely diffusible molecule selectively activates AMPA receptors in a light-dependent fashion. It primarily acts on amacrine and retinal ganglion cells, although a minor effect on bipolar cells has been observed. As such, it complements previous pharmacological approaches based on photochromic channel blockers and increases the potential of photopharmacology in vision restoration.

A red-shifted photochromic sulfonylurea for the remote control of pancreatic beta cell function.

Azobenzene photoresponsive elements can be installed on sulfonylureas, yielding optical control over pancreatic beta cell function and insulin release. An obstacle to such photopharmacological approaches remains the use of ultraviolet-blue illumination. Herein, we synthesize and test a novel yellow light-activated sulfonylurea based on a heterocyclic azobenzene bearing a push-pull system.

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools.

Optogenetic tools have become indispensable in neuroscience to stimulate or inhibit excitable cells by light. Channelrhodopsin-2 (ChR2) variants have been established by mutating the opsin backbone or by mining related algal genomes. As an alternative strategy, we surveyed synthetic retinal analogues combined with microbial rhodopsins for functional and spectral properties, capitalizing on assays in C. elegans, HEK cells and larval Drosophila. Compared with all-trans retinal (ATR), Dimethylamino-retinal (DMAR) shifts the action spectra maxima of ChR2 variants H134R and H134R/T159C from 480 to 520 nm. Moreover, DMAR decelerates the photocycle of ChR2(H134R) and (H134R/T159C), thereby reducing the light intensity required for persistent channel activation. In hyperpolarizing archaerhodopsin-3 and Mac, naphthyl-retinal and thiophene-retinal support activity alike ATR, yet at altered peak wavelengths. Our experiments enable applications of retinal analogues in colour tuning and altering photocycle characteristics of optogenetic tools, thereby increasing the operational light sensitivity of existing cell lines or transgenic animals.

Long wavelength optical control of glutamate receptor ion channels using a tetra-ortho-substituted azobenzene derivative.

A tetra-ortho-chloro substituted azobenzene unit was incorporated into a photoswitchable tethered ligand for ionotropic glutamate receptors. This compound confers the modified protein with the unusual optical responses of the substituted azo scaffold permitting channel opening with yellow and red light and channel closing with blue light.

Dyspraxia, motor function and visual-motor integration in autism.

This project assessed dyspraxia in high-functioning school aged children with autism with a focus on Ideational Praxis. We examined the association of specific underlying motor function including eye movement with ideational dyspraxia (sequences of skilled movements) as well as the possible role of visual-motor integration in dyspraxia. We found that compared to IQ-, sex- and age-matched typically developing children, the children with autism performed significantly worse on: Ideational and Buccofacial praxis; a broad range of motor tests, including measures of simple motor skill, timing and accuracy of saccadic eye movements and motor coordination; and tests of visual-motor integration. Impairments in individual children with autism were heterogeneous in nature, although when we examined the praxis data as a function of a qualitative measure representing motor timing, we found that children with poor motor timing performed worse on all praxis categories and had slower and less accurate eye movements while those with regular timing performed as well as typical children on those same tasks. Our data provide evidence that both motor function and visual-motor integration contribute to dyspraxia. We suggest that dyspraxia in autism involves cerebellar mechanisms of movement control and the integration of these mechanisms with cortical networks implicated in praxis.

Oral motor dysfunction and feeding difficulties in nephropathic cystinosis.

Nephropathic cystinosis is a genetic disorder in which the amino acid cystine accumulates in lysosomes, resulting in multiorgan dysfunction. Progressive neuromuscular dysfunction, with bulbar and upper extremity weakness, has been described in adults with this disorder. The purpose of the present study was to determine whether there was evidence of early bulbar involvement, suggested by feeding difficulties or oral motor dysfunction in these patients, and whether the feeding and oral motor problems were associated with other evidence of neurologic dysfunction. Twenty-two children and adolescents with nephropathic cystinosis were studied. Parents completed questionnaires on feeding history and oral motor problems. Eighteen patients were given an oral motor examination, and 14 received a complete neurologic examination. The majority of children had a history of feeding difficulties. Seven children required a gastrostomy tube. Abnormalities on oral motor examination included hypotonia, abnormal gag reflex, and throaty or congested voice. Abnormalities on neurologic examination included hypotonia, muscle weakness, gross and fine motor dysfunction, and ataxia. The results indicate that feeding difficulties and oral motor dysfunction are common in children with cystinosis and appear to correlate with the general degree of neurologic dysfunction. Long-term follow-up is necessary to determine whether the early oral motor problems predict the later development of the progressive myopathy observed in adults with cystinosis.

Behavioural profiles of children and adolescents after pre- or perinatal unilateral brain damage.

Recent case reports of individuals with early-onset damage to the prefrontal cortex have suggested that such early insults could result in severely impaired social behaviour in later childhood and adolescence. The investigators speculated that the acquisition of complex social conventions and moral rules had been impaired. In a large cohort of children, we sought to determine whether early focal brain insults might result in clinically significant behavioural or emotional problems. This study reports on 39 children with pre- or perinatal-onset unilateral brain damage (focal lesion) from cerebral infarction or intraparenchymal haemorrhage, using the Achenbach Child Behavior Checklist to assess the presence or absence of behavioural and emotional difficulties. Two-thirds of the subjects had left hemisphere (LH) lesions and one-third had right hemisphere (RH) lesions. Age range was 4.0-15.4 years at the time of questionnaire completion. Their results were compared with those of 54 control children. Analyses were conducted on focal lesion versus controls, RH versus LH lesion, frontal versus non-frontal lesion, and seizure versus non-seizure groups. When the effect of IQ was partialled out, there were no significant differences on the nine Behavior Problem scales, the Internalizing-Externalizing dichotomy or the Total Problem score for any of the group comparisons. Our subjects showed no evidence of clinically significant behavioural or emotional problems, even when the frontal lobe was involved. Individuals with more extensive and bilateral damage may be at higher risk of significant behavioural and emotional dysfunction than were those in our study population. In future studies of brain-behaviour relationships in developing children, all potential causes for any observed behavioural abnormalities, such as genetic and environmental factors and toxin exposure, must be considered before concluding that specific anatomical lesions are causally related to specific behavioural outcomes.

"Motor" impairment in Asperger syndrome: evidence for a deficit in proprioception.

Motor impairment has frequently been described in Asperger syndrome (AS), a pervasive developmental disorder included in the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV). Previous research focusing on this motor dysfunction has yielded inconsistent results, and the "clumsiness" observed clinically remains poorly defined. To clarify further the issue of motor impairment, we compared a group of 10 children and young adults who met DSM-IV criteria for AS with a control group with no neurological impairment. Subjects were matched on age, sex, socioeconomic status, and Verbal IQ. A broad battery of motoric tests was administered. Subjects with AS were found to perform more poorly than controls on tests of apraxia, one-leg balance with eyes closed, tandem gait, and repetitive finger-thumb apposition. No significant differences were found on tests of finger tapping, grooved pegboard, trail making, or visual-motor integration. The pattern of impairments suggests that a proprioceptive deficit may underlie the incoordination observed in AS and that these individuals may be overreliant on visual input to maintain balance and position in space.

Sensitivity and specificity of a computerized test of attention in the diagnosis of Attention-Deficit/Hyperactivity Disorder.

Attention-Deficit/Hyperactivity Disorder (ADHD) is difficult to diagnose due to the subjectivity of its symptoms and lack of specific assessment measures. Computerized tests of attention have recently been used as objective measures that may assist in the diagnosis of the disorder. The present study evaluated consistency between the Conners Parent Rating Scale and the Test of Variables of Attention (TOVA), which is a computerized test of attention designed to identify symptoms associated with ADHD, in children clinically diagnosed with ADHD (n = 28) and controls (n = 20). Our results showed that both the Conners and the TOVA indicated significant problem areas suggestive of an attention deficit in approximately 85% of children who were clinically diagnosed with ADHD. However, the TOVA also found attentional problems in approximately 30% of control children, whereas none of the controls scored abnormally on the Conners. As computerized measures are administered more frequently, there may be a risk of overdiagnosis and treatment of "ADHD" in normal children. A combined approach using questionnaires, clinical evaluation, and computerized tests of attention in the assessment of possible ADHD may provide the most accurate means of diagnosis.

Development of perceptual asymmetry for free viewing of chimeric stimuli.

Free-viewing chimeric stimuli tasks have been used in a number of studies to assess perceptual asymmetries and draw inferences about hemispheric lateralization in children and adults. In order to determine whether perceptual asymmetries for nonverbal information are present in children, a free-viewing chimeric stimuli task was used in 63 normally developing 6- through 16-year-old children. Stimuli included affect (happy faces), gender, quantity, and shape. An overall left hemispace (LHS) advantage was present by 6 years of age. This LHS preference was more prominent by age 10 and then plateaued. No preference for shape was detected at any of the age ranges studied. These results suggest that perceptual asymmetries for visual stimuli develop during childhood and appear to reach a plateau by age 10. The observed specificity for certain types of nonverbal stimuli should be taken into account in future studies of perceptual asymmetry in both normal and neurologically impaired children.

Regional size reduction in the human corpus callosum following pre- and perinatal brain injury.

This morphometric study examined two aspects of corpus callosum development: pediatric cortico-callosal topography and developmental neuroplasticity subsequent to perinatal brain injury. In vivo magnetic resonance imaging was used to quantify the total midsagittal cross-sectional area and five anterioposterior subregions of the callosum in 10 children with focal lesions and 86 healthy volunteer control subjects. Nine of the ten children with early injury showed a reduction in the total area of the callosum relative to matched controls. The area of the total callosum cross-section was inversely proportional to the size of lesion. All patients displayed region-specific size reduction. This regional thinning bore a topographical relationship to the lesion sites. Reduction in anterior subregions 1, 2 and 3 was respectively associated with lesions in the anterior inferior frontal area, the middle and superior frontal region, and the precentral area. Attenuation of subregion 4 corresponded to anterior parietal lesions, and thinning of subregion 5 occurred with posterior parietal injury. This cortical-callosal pattern coincides with adult and nonhuman primate mappings. Callosal thinning despite the early onset of the lesions suggests limits to developmental neuroplasticity.

Neurological and MRI profiles of children with developmental language impairment.

Children with developmental language impairment (LI) are defined partly by the absence of other identifiable neurological diagnoses. Such children are generally considered to be neurologically normal, but no systematic studies of neurological function have been reported. We obtained detailed medical histories and conducted neurological examinations for 72 children aged 5 to 14 years with LI and 82 typically developing age-matched control children. All the children took a standardized test of language, and those who were at least 8 years old and were willing to have brain MRI scans (35 children with LI and 27 control children) had scans. Analysis of developmental milestones from the medical histories revealed that children with LI were not only significantly later in speaking, but also mildly but significantly delayed in motor milestones, particularly walking. On neurological examination, abnormalities were found in 70% of the children with LI and only 22% of the control children. The most common abnormalities in the LI group included obligatory synkinesis, fine motor impairments, and hyperreflexia. The children with LI with the most abnormal neurological findings had the lowest language scores. Finally, 12 of 35 children with LI had abnormalities on their MRI scan, while none of the 27 control children had abnormal scans. Abnormal findings included ventricular enlargement (in five), central volume loss (in three), and white matter abnormalities (in four). These findings suggest that developmental LI is not an isolated finding but is indicative of more widespread nervous system dysfunction. Children with LI may need more comprehensive intervention programs than language therapy alone, depending on their other areas of dysfunction. Early identification of such problems may allow for more successful remediation.

A hierarchical analysis of block design errors in children with early focal brain damage.

This study investigated the differential effects of very early damage to the left hemisphere (LH) or right hemisphere (RH) on visuospatial processing. Twenty-two children who had suffered either LH or RH strokes in the pre- or perinatal period were included in the study. The Block Design subtest of the Wechsler Intelligence Scale for Children-Revised (Wechsler, 1974) was used. Each missed item was coded as either a global error (e.g., broken configuration), local error (e.g., incorrect details), or time fail error (i.e., not completed within the allotted time). Results showed that the LH lesion and RH lesion groups had similar full scale IQs, verbal IQs, and performance IQs and were within the average to low average range. Block Design scaled scores were also within the average to low average range and did not significantly differ between the 2 lesion groups. Error analysis revealed, however, that the RH focal lesion group produced a significantly higher percentage of global errors than did the LH lesion group, whereas the LH lesion group produced a significantly higher percentage of local errors than did the RH lesion group. The groups did not differ on their percentage of time fail errors. These results are consistent with previous findings that suggest that the RH is involved in more global aspects of visual processing, whereas the LH mediates the more detailed, local aspects of visual information. The fact that these differences in processing are present after such early focal damage implies that hemispheric specialization for visuospatial processing occurs very early in brain development.

Neurobehavioral consequences of a genetic metabolic disorder: visual processing deficits in infantile nephropathic cystinosis.

OBJECTIVE: The purpose of the current study was to further characterize the nature of the visual processing deficit in infantile nephropathic cystinosis. It was hypothesized that children with cystinosis would demonstrate a dissociation between visuospatial and visuoperceptual abilities, with impaired spatial functioning and intact perceptual functioning. Hypotheses were based on cognitive studies to date as well as on a review of the visual processing literature. BACKGROUND: Infantile nephropathic cystinosis is a genetic metabolic disorder in which the amino acid cystine accumulates in various organs, including the kidney, cornea, thyroid, and brain. The existing neurocognitive literature suggests the presence of a visual processing deficit against a background of generally normal intellectual capacity. The nature of the deficit, however, is still somewhat ambiguous. METHOD: Study participants were 141 children (33 with cystinosis and 108 controls), ages 5 through 14 years. Tests of visuospatial and visuoperceptual functioning were administered. RESULTS: Data were analyzed using hierarchical regression analyses and MANCOVA. After covarying for relevant demographic variables, the cystinosis group consistently demonstrated impairments in spatial processing, whereas perceptual processing was largely intact. CONCLUSIONS: Results support the hypothesis of a dissociation in visual processing. Findings suggest that cystinosis has a differential effect on the two cortical visual processing streams, with spatial functions affected to a greater extent than perceptual functions. The present study has implications for brain-behavior relationships in other genetic disorders as well.

Total Synthesis of (+)-Halichlorine: An Inhibitor of VCAM-1 Expression.

The diastereoselective addition of the highly functionalized organozinc compound 1 to the aldehyde 2 in the presence of the chiral amino alcohol 3 (-->4) is a key step in the first total synthesis of (+)-halichlorine. A series of protections/deprotections and a macrolaconization complete the synthesis. Halichlorine selectively inhibits the expression of the cell adhesion molecule VCAM-1. TBS=tert-butyldimethylsilyl.