Tatsuo Mano

Regulation of Dendritogenesis via a Lipid-Raft-Associated Ca2+/Calmodulin-Dependent Protein Kinase CLICK-III/CaMKIγ

Ca(2+) signaling plays a central role in activity-dependent regulation of dendritic arborization, but key molecular mechanisms downstream of calcium elevation remain poorly understood. Here we show that the C-terminal region of the Ca(2+)/calmodulin-dependent protein kinase CLICK-III (CL3)/CaMKIgamma, a membrane-anchored CaMK, was uniquely modified by two sequential lipidification steps: prenylation followed by a kinase-activity-regulated palmitoylation. These modifications were essential for CL3 membrane anchoring and targeting into detergent-resistant lipid microdomains (or rafts) in the dendrites. We found that CL3 critically contributed to BDNF-stimulated dendritic growth. Raft insertion of CL3 specifically promoted dendritogenesis of cortical neurons by acting upstream of RacGEF STEF and Rac, both present in lipid rafts. Thus, CL3 may represent a key element in the Ca(2+)-dependent and lipid-raft-delineated switch that turns on extrinsic activity-regulated dendrite formation in developing cortical neurons.

Control of Cortical Axon Elongation by a GABA-Driven Ca2+/Calmodulin-Dependent Protein Kinase Cascade

Ca2+ signaling plays important roles during both axonal and dendritic growth. Yet whether and how Ca2+ rises may trigger and contribute to the development of long-range cortical connections remains mostly unknown. Here, we demonstrate that two separate limbs of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)–CaMKI cascades, CaMKK–CaMKIα and CaMKK–CaMKIγ, critically coordinate axonal and dendritic morphogenesis of cortical neurons, respectively. The axon-specific morphological phenotype required a diffuse cytoplasmic localization and a strikingly α-isoform-specific kinase activity of CaMKI. Unexpectedly, treatment with muscimol, a GABAA receptor agonist, selectively stimulated elongation of axons but not of dendrites, and the CaMKK–CaMKIα cascade critically mediated this axonogenic effect. Consistent with these findings, during early brain development, in vivo knockdown of CaMKIα significantly impaired the terminal axonal extension and thereby perturbed the refinement of the interhemispheric callosal projections into the contralateral cortices. Our findings thus indicate a novel role for the GABA-driven CaMKK–CaMKIα cascade as a mechanism critical for accurate cortical axon pathfinding, an essential process that may contribute to fine-tuning the formation of interhemispheric connectivity during the perinatal development of the CNS.

Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

Histone deacetylases (HDACs) are potential therapeutic targets of polyglutamine (pQ) diseases including Huntington’s disease (HD) that may function to correct aberrant transcriptional deactivation caused by mutant pQ proteins. HDAC3 is a unique class 1 HDAC found in both the cytoplasm and in the nucleus. However, the precise functions of HDAC3 in the two cellular compartments are only vaguely known. HDAC3 directly binds to huntingtin (Htt) with short pQ and this interaction is important for suppressing neurotoxicity induced by HDAC3. With long pQ Htt, the interaction with HDAC3 is inhibited, and this supposedly promotes neuronal death, indicating that HDAC3 would be a good therapeutic target for HD. However, the knockout of one HDAC3 allele did not show any efficacy in reducing neurodegenerative symptoms in a mouse model of HD. Therefore, the role of HDAC3 in the pathogenesis of HD has yet to be fully elucidated. We attempted to resolve this issue by focusing on the different roles of HDAC3 on cytoplasmic and nuclear Htt aggregates. In addition to supporting the previous findings, we found that HDAC3 preferentially binds to nuclear Htt over cytoplasmic ones. Specific HDAC3 inhibitors increased the total amount of Htt aggregates by increasing the amount of nuclear aggregates. Both cytoplasmic and nuclear Htt aggregates were able to suppress endogenous HDAC3 activity, which led to decreased nuclear proteasome activity. Therefore, we concluded that Htt aggregates impair nuclear proteasome activity through the inhibition of HDAC3. Our findings provide new insights regarding cross-compartment proteasome regulation.

Neuron-specific methylome analysis reveals epigenetic regulation and tau-related dysfunction of BRCA1 in Alzheimer’s disease

Significance To extract critical information from Alzheimer’s disease (AD) postmortem brains that may otherwise be lost, we chose to screen epigenetic signatures. Epigenome analysis is a robust methodology in terms of its cell type and gene specificity, suitability for high-throughput analysis, and resistance to postmortem degradation. Analysis of the neuron-specific methylome revealed a variety of differentially methylated genes, including BRCA1. We demonstrate the pathogenic relevance of compromised genomic integrity by analyzing the neuroprotective function of BRCA1 against amyloid β (Aβ)-induced DNA double-strand breaks. Furthermore, insolubility of BRCA1 under the presence of aggregated tau suggested the reason for its dysfunction despite enhanced expression. We provide insight into the pathomechanism of AD and demonstrate the potential of screening neuron-specific methylome to reveal new pathogenic contributors. Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by pathology of accumulated amyloid β (Aβ) and phosphorylated tau proteins in the brain. Postmortem degradation and cellular complexity within the brain have limited approaches to molecularly define the causal relationship between pathological features and neuronal dysfunction in AD. To overcome these limitations, we analyzed the neuron-specific DNA methylome of postmortem brain samples from AD patients, which allowed differentially hypomethylated region of the BRCA1 promoter to be identified. Expression of BRCA1 was significantly up-regulated in AD brains, consistent with its hypomethylation. BRCA1 protein levels were also elevated in response to DNA damage induced by Aβ. BRCA1 became mislocalized to the cytoplasm and highly insoluble in a tau-dependent manner, resulting in DNA fragmentation in both in vitro cellular and in vivo mouse models. BRCA1 dysfunction under Aβ burden is consistent with concomitant deterioration of genomic integrity and synaptic plasticity. The Brca1 promoter region of AD model mice brain was similarly hypomethylated, indicating an epigenetic mechanism underlying BRCA1 regulation in AD. Our results suggest deterioration of DNA integrity as a central contributing factor in AD pathogenesis. Moreover, these data demonstrate the technical feasibility of using neuron-specific DNA methylome analysis to facilitate discovery of etiological candidates in sporadic neurodegenerative diseases.

Tacrolimus-Induced Reversible Cerebral Vasoconstriction Syndrome with Delayed Multi-Segmental Vasoconstriction

Reversible cerebral vasoconstriction syndrome (RCVS) is a cerebrovascular syndrome characterized by multi-segmental constrictions of the cerebral arteries that resolves spontaneously within 3 months. Although RCVS is considered to be due to transient dysregulation of vascular tone, the exact pathomechanism remains unclear. We describe the case of a 15-year-old girl with RCVS induced by tacrolimus, who developed generalized seizure during the postoperative course of orthotropic heart transplantation. Magnetic resonance imaging at symptom onset showed a few vasoconstrictions accompanying brain edema and convexity subarachnoid hemorrhage. Although her neurological conditions rapidly improved after discontinuing tacrolimus, a repeat magnetic resonance angiogram demonstrated delayed progression of the multi-segmental vasoconstrictions followed by subsequent resolution. Our case demonstrates that cautious observation of the cerebral arteries using magnetic resonance angiography and careful management of vasoconstrictions with vasodilators are necessary for delayed vasoconstrictions even when the clinical symptoms improve.

An Autopsy Case of Familial Neuronal Intranuclear Inclusion Disease with Dementia and Neuropathy

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disease with marked variety in its clinical manifestations. While characteristic neuroimaging and skin biopsy findings are important clues to the diagnosis, autopsy studies are still important for confirming the exact disease features. We herein report the case of a patient who received an antemortem diagnosis of familial NIID with dementia-dominant phenotype that was later confirmed by an autopsy. Our report is the first to document a case of autopsy-confirmed NIID involving both cognitive impairment and sensorimotor neuropathy.

DNA methylation level of the neprilysin promoter in Alzheimer's disease brains

Neprilysin (NEP), a membrane-bound metalloprotease, has been shown to play an essential role in the clearance of amyloid beta (Aβ) peptides. Previous studies have reported that NEP expression is downregulated in the normal aging brain as well as in the Alzheimers disease (AD) brain, providing evidence that the downregulation of NEP expression contributes to the age-dependent deposition of Aβ-containing plaques, a pathological hallmark of AD. However, the mechanisms underlying the downregulation remain unclear. In this study, we explored the relationship between DNA methylation status of CpG islands in the NEP promoter and its expression level in AD brains. We performed pyrosequencing analyses to detect the DNA methylation level in 31 postmortem AD brains and 40 normal control brains. All 30 CpG sites showed no clear difference in methylation level. To further focus on methylation changes specific to neuronal cells, we performed methylation array experiments using neuronal nuclei from postmortem brains and found no clear difference in the methylation level between AD and normal control samples. Our detailed analyses, with a substantial number of brain samples, provide the first convincing evidence that DNA methylation of the NEP promoter is not involved in AD development and progression.

Longitudinally extensive vasogenic edema following spinal cord infarction

Although transient lesion expansion has been reported after spinal cord infarction, longitudinally extensive lesion expanding more than several vertebral segments is extremely rare. We report a 45‐year‐old man having spinal cord infarction with subsequent longitudinally extensive lesion expansion on magnetic resonance imaging (MRI) 10 days after the infarction. The length of T2 hyperintense lesion was four vertebral segments on initial MRI but enlarged to span 12 vertebral segments on subsequent MRI, with Lhermittes sign, but otherwise without neurological deterioration. The expanded lesion extended beyond the anterior spinal artery region with high apparent diffusion coefficient value in the acute phase but improved within 10 days after starting cervical collar, suggesting that lesion expansion was due to vasogenic edema. This case illustrates that transient longitudinal lesion expansion accompanied by spinal cord infarction can reach lengths of up to 12 vertebral segments. Caution should be exercised while interpreting MRI findings to avoid excessive treatment or secondary damage.

Isolated unilateral ptosis due to midbrain infarction

A 74-year-old man with a history of diabetes mellitus, dyslipidemia, and hypertension presented with sudden left ptosis. Neurological examination revealed no apparent ophthalmoplegia (Fig. 1a). Pupils were equal with normal pupillary light reflex and near reaction. Facial sweating was preserved. Subsequent slight diplopia was noted on rightward, upward, and downward gaze. Hess charts were compatible with slight left oculomotor palsy (Fig. 1b). Brain MRI showed small left paramedian midbrain infarction (Fig. 1c, d). Atherosclerotic change in the basilar artery was observed without significant stenosis of basilar or left posterior cerebral artery (Fig. 1e). Atherothrombotic or lacunar stroke of the anteromedial midbrain artery was suspected. Left ptosis gradually improved in two months. The levator palpebrae superioris muscles are innervated by the central caudal subnucleus of oculomotor nucleus. Despite the bilateral organization of this subnucleus, damage to the paramedian fascicle originating from this subnucleus can cause isolated unilateral ptosis with minimal extraocular involvement. Isolated unilateral ptosis due to ischemia is a clinical pitfall, and brain MRI should be considered.

Severe visual impairment and subclinical encephalitis preceding clinical signs of chondritis in relapsing polychondritis

A 71‐year‐old woman with a 6‐month history of relapsing bilateral anterior scleritis presented with severe right visual impairment due to posterior scleritis. Despite radiological signs of encephalitis, the patient and her family members noticed no cognitive decline. The patient subsequently developed slight auricular pain without any visual changes such as redness or swelling, which, however, showed increased uptake of 18F‐fluorodeoxyglucose on positron emission tomography. Auricular cartilage biopsy revealed perichondrial inflammation suggesting relapsing polychondritis. Steroid therapy improved her symptoms and radiological findings. This case illustrates that asymptomatic brain inflammatory lesions can precede clinical signs of chondritis in relapsing polychondritis, and that auricular cartilage biopsy should be considered even with mild auricular pain without apparent clinical findings of inflammation.