Neuronal Ceroid Lipofuscinosis Type 6 (CLN6) clinical findings and molecular diagnosis: Costa Rica's experience.

BACKGROUND: Commonly known as Batten disease, the neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of rare pediatric lysosomal storage disorders characterized by the intracellular accumulation of autofluorescent material (known as lipofuscin), progressive neurodegeneration, and neurological symptoms. In 2002, a disease-causing NCL mutation in the CLN6 gene was identified (c.214G > T) in the Costa Rican population, but the frequency of this mutation among local Batten disease patients remains incompletely characterized, as do clinical and demographic attributes for this rare patient population. OBJECTIVE: To describe the main sociodemographic and clinical characteristics of patients with a clinical diagnosis for Batten Disease treated at the National Children's Hospital in Costa Rica and to characterize via molecular testing their causative mutations. METHODS: DNA extracted from buccal swabs was used for CLN6 gene sequencing. Participants' sociodemographic and clinical characteristics were also obtained from their medical records. RESULTS: Nine patients with a clinical diagnosis of Batten disease were identified. Genetic sequencing determined the presence of the previously described Costa Rican homozygous mutation in 8 of 9 cases. One patient did not have mutations in the CLN6 gene. In all cases where the Costa Rican CLN6 mutation was present, it was accompanied by a substitution in intron 2. Patients were born in 4 of the 7 Costa Rican provinces, with an average onset of symptoms close to 4 years of age. No parental consanguinity was present in pedigrees. Initial clinical manifestations varied between patients but generally included: gait disturbances, language problems, visual impairment, seizures and psychomotor regression. Cortical and cerebellar atrophy was a constant finding when neuroimaging was performed. Seizure medication was a common element of treatment regimens. CONCLUSIONS: This investigation supports that the previously characterized c.214G > T mutation is the most common causative NCL mutation in the Costa Rican population. This mutation is geographically widespread among Costa Rican NCL patients and yields a clinical presentation similar to that observed for CLN6 NCL patients in other geographies.

MARCKS regulates neuritogenesis and interacts with a CDC42 signaling network.

Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks-/- cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.

MARCKS Is Necessary for Netrin-DCC Signaling and Corpus Callosum Formation.

Axons of the corpus callosum (CC), the white matter tract that connects the left and right hemispheres of the brain, receive instruction from a number of chemoattractant and chemorepulsant cues during their initial navigation towards and across the midline. While it has long been known that the CC is malformed in the absence of Myristoylated alanine-rich C-kinase substrate (MARCKS), evidence for a direct role of MARCKS in axon navigation has been lacking. Here, we show that MARCKS is necessary for Netrin-1 (NTN1) signaling through the DCC receptor, which is critical for axon guidance decisions. Marcks null (Marcks-/-) neurons fail to respond to exogenous NTN1 and are deficient in markers of DCC activation. Without MARCKS, the subcellular distributions of two critical mediators of NTN1-DCC signaling, the tyrosine kinases PTK2 and SRC, are disrupted. Together, this work establishes a novel role for MARCKS in axon dynamics and highlights the necessity of MARCKS as an organizer of DCC signaling at the membrane.

Acute Cytotoxic and Vasogenic Edema after Subarachnoid Hemorrhage: A Quantitative MRI Study.

BACKGROUND AND PURPOSE: The mechanism of early brain injury following subarachnoid hemorrhage is not well understood. We aimed to evaluate if cytotoxic and vasogenic edema are contributing factors. MATERIALS AND METHODS: A retrospective analysis was conducted in patients with SAH undergoing diffusion-weighted MR imaging within 72 hours of onset. Apparent diffusion coefficient values derived from DWI were evaluated by using whole-brain histograms and 19 prespecified ROIs in patients with SAH and controls with normal findings on MRI. Cytotoxic edema observed outside the ROIs was assessed in patients with SAH. The average median ADC values were compared between patients with SAH and controls and patients with SAH with mild (Hunt and Hess 1-3) versus severe early brain injury (Hunt and Hess 4-5). RESULTS: We enrolled 33 patients with SAH and 66 controls. The overall average median whole-brain ADC was greater for patients with SAH (808 × 10-6 mm2/s) compared with controls (788 × 10-6 mm2/s, P < .001) and was higher in patients with SAH across ROIs after adjusting for age: cerebral gray matter (826 versus 803 × 10-6 mm2/s, P = .059), cerebral white matter (793 versus 758 × 10-6 mm2/s, P = .023), white matter tracts (797 versus 739 × 10-6 mm2/s, P < .001), and deep gray matter (754 versus 713 × 10-6 mm2/s, P = .016). ADC values trended higher in patients with Hunt and Hess 4-5 versus those with Hunt and Hess 1-3. Early cytotoxic edema was observed in 13 (39%) patients with SAH and was more prevalent in those with severe early brain injury (87.5% of patients with Hunt and Hess 4-5 versus 24.0% of those with Hunt and Hess 1-3, P = .001). CONCLUSIONS: Age-adjusted ADC values were globally increased in patients with SAH compared with controls, even in normal-appearing brain regions, suggesting diffuse vasogenic edema. Cytotoxic edema was also present in patients with SAH and correlated with more severe early brain injury.

Clathrin assembly protein AP-2 is detected in both neurons and glia, and its reduction is prominent in layer II of frontal cortex in Alzheimer's disease.

There are several adaptor proteins associated with clathrin coated vesicles. Among them are AP180 and AP-2. We and others have previously described synaptic localization of AP180. AP180 immunoreactivity is altered in both the superior frontal gyrus and hippocampus in Alzheimer's disease (AD). We here investigate the location and alteration of another adaptor protein, AP-2. In contrast to AP180, we have found that AP-2 is expressed by both neurons and glia. Furthermore, the only noticeable change of AP-2 in AD is a loss of its immunoreactivity in layer II of the superior frontal gyrus.

Expression profiles of multiple genes in single neurons of Alzheimer's disease.

Many changes have been described in the brains of Alzheimer's disease (AD) patients, including loss of neurons and formation of senile plaques and neurofibrillary tangles. The molecular mechanisms underlying these pathologies are unclear. Northern blot, dot-blot, and reverse transcription-coupled PCR analyses have demonstrated altered expression levels of multiple messages in AD brain. Because not all cells are equally affected by the disease, these methods obviously cannot study the changes in relation to disease states of individual cells. We address this problem by using antisense RNA profiling of single cells. We present expression profiles of single neurons at early and late stages of AD and describe statistical tools for data analysis. With multivariate canonical analysis, we were able to distinguish the disease state on the basis of altered expression of multiple messages. To validate this approach, we compared results obtained by this approach with results obtained by in situ hybridization analysis. When the neurofilament medium subunit was used as a marker, our results from an antisense RNA profiling revealed no change in neurofilament medium subunit expression between early- and late-stage AD, consistent with findings obtained with in situ hybridization. However, our results obtained by either analysis at the single-cell level differed from the reported decrease in AD neocortex obtained by Northern blot analysis [Kittur, S., Hoh, J., Endo, H., Tourtellotte, W., Weeks, B. S., Markesbery, W. & Adler, W. (1994) J. Geriatr. Psychiatry Neurol. 7, 153-158]. Thus, the strategy of using the single-cell antisense RNA approach to identify altered gene expression in postmortem AD brain, followed by detailed in situ hybridization studies for genes of interest, is valuable in the study of the molecular mechanisms underlying AD neuropathology.