Altered thalamocortical tract trajectory growth with undisrupted thalamic parcellation pattern in human lissencephaly brain at mid-gestational stage.

Proper topographically organized neural connections between the thalamus and the cerebral cortex are mandatory for thalamus function. Thalamocortical (TC) fiber growth begins during the embryonic period and completes by the third trimester of gestation, so that human neonates at birth have a thalamus with a near-facsimile of adult functional parcellation. Whether congenital neocortical anomaly (e.g., lissencephaly) affects TC connection in humans is unknown. Here, via diffusion MRI fiber-tractography analysis of long-term formalin-fixed postmortem fetal brain diagnosed as lissencephaly in comparison with an age-matched normal one, we found similar topological patterns of thalamic subregions and of internal capsule parcellated by TC fibers. However, lissencephaly fetal brain showed white matter structural changes, including fewer/less organized TC fibers and optic radiations, and much less cortical plate invasion by TC fibers - particularly around the shallow central sulcus. Diffusion MRI fiber tractography of normal fetal brains at 15, 23, and 26 gestational weeks (GW) revealed dynamic volumetric change of each parcellated thalamic subregion, suggesting coupled developmental progress of the thalamus with the corresponding cortex. Moreover, from GW23 and GW26 normal fetal brains, TC endings in the cortical plate could be delineated to reflect cumulative progressive TC invasion of cortical plate. By contrast, lissencephaly brain showed a dramatic decrease in TC invasion of the cortical plate. Our study thus shows the feasibility of diffusion MRI fiber tractography in postmortem long-term formalin-fixed fetal brains to disclose the developmental progress of TC tracts coordinating with thalamic and neocortical growth both in normal and lissencephaly fetal brains at mid-gestational stage.

Revealing intact neuronal circuitry in centimeter-sized formalin-fixed paraffin-embedded brain.

Tissue-clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.

Diagnostic Challenges of Neuromuscular Disorders after Whole Exome Sequencing.

BACKGROUND: Whole-exome sequencing (WES) facilitates the diagnosis of hereditary neuromuscular disorders. To achieve an accurate diagnosis, physicians should interpret the genetic report carefully along with clinical information and examinations. We described our experience with (1) clinical validation in patients with variants found using WES and (2) a diagnostic approach for those with negative findings from WES. METHODS: WES was performed on patients with the clinical impression of hereditary neuromuscular disorders. Information on clinical manifestations, neurological examination, electrodiagnostic studies, histopathology of muscle and nerve, and laboratory tests were collected. RESULTS: Forty-one patients (Male/Female: 18/23, age of onset: 34.5±15.9) accepted WES and were categorized into four scenarios: (1) patients with a positive WES result, (2) patients with an inconclusive WES result but supporting clinical data, (3) negative findings from WES, but a final diagnosis after further work-up, and (4) undetermined etiology from WES and in further work-ups. The yield rate of the initial WES was 63.4% (26/41). Among these, seventeen patients had positive WES result, while the other nine patients had inconclusive WES result but supporting clinical data. Notably, in the fifteen patients with negative findings from WES, four patients (26.7%) achieved a diagnosis after further workup: tumor-induced osteomalacia, metabolic myopathy with pathogenic variants in mitochondrial DNA, microsatellite expansion disease, and vasculitis-related neuropathy. The etiologies remained undetermined in eleven patients (myopathy: 7, neuropathy: 4) after WES and further workup. CONCLUSIONS: It is essential to design genotype-guided molecular studies to correlate the identified variants with their clinical features. For patients who had negative findings from WES, acquired diseases, mitochondrial DNA disorders and microsatellite expansion diseases should be considered.

ARMS-NF-κB signaling regulates intracellular ROS to induce autophagy-associated cell death upon oxidative stress.

Ankyrin repeat-rich membrane spanning (ARMS) plays roles in neural development, neuropathies, and tumor formation. Such pleiotropic function of ARMS is often attributed to diverse ARMS-interacting molecules in different cell context. However, it might be achieved by ARMS' effect on global biological mediator like reactive oxygen species (ROS). We established ARMS-knockdown in melanoma cells (siARMS) and in Drosophila eyes (GMR>dARMS RNAi ) and challenged them with H2O2. Decreased ARMS in both systems compromises nuclear translocation of NF-κB and induces ROS, which in turn augments autophagy flux and confers susceptibility to H2O2-triggered autophagic cell death. Resuming NF-κB activity or reducing ROS by antioxidants in siARMS cells and GMR>dARMS RNAi fly decreases intracellular peroxides level concurrent with reduced autophagy and attenuated cell death. Conversely, blocking NF-κB activity in wild-type flies/melanoma enhances ROS and induces autophagy with cell death. We thus uncover intracellular ROS modulated by ARMS-NFκB signaling primes autophagy for autophagic cell death upon oxidative stress.

Involvement of a BH3-only apoptosis sensitizer gene Blm-s in hippocampus-mediated mood control.

Mood disorders are an important public health issue and recent advances in genomic studies have indicated that molecules involved in neurodevelopment are causally related to mood disorders. BLM-s (BCL-2-like molecule, small transcript isoform), a BH3-only proapoptotic BCL-2 family member, mediates apoptosis of postmitotic immature neurons during embryonic cortical development, but its role in the adult brain is unknown. To better understand the physiological role of Blm-s gene in vivo, we generated a Blm-s-knockout (Blm-s-/-) mouse. The Blm-s-/- mice breed normally and exhibit grossly normal development. However, global depletion of Blm-s is highly associated with depression- and anxiety-related behaviors in adult mutant mice with intact learning and memory capacity. Functional magnetic resonance imaging of adult Blm-s-/- mice reveals reduced connectivity mainly in the ventral dentate gyrus (vDG) of the hippocampus with no alteration in the dorsal DG connectivity and in total hippocampal volume. At the cellular level, BLM-s is expressed in DG granule cells (GCs), and Blm-s-/- mice show reduced dendritic complexity and decreased spine density in mature GCs. Electrophysiology study uncovers that mature vGCs in adult Blm-s-/- DG are intrinsically more excitable. Interestingly, certain genetic variants of the human Blm homologue gene (VPS50) are significantly associated with depression traits from publicly resourced UK Biobank data. Taken together, BLM-s is required for the hippocampal mood control function. Loss of BLM-s causes abnormality in the electrophysiology and morphology of GCs and a disrupted vDG neural network, which could underlie Blm-s-null-associated anxiety and depression.

RTL1/PEG11 imprinted in human and mouse brain mediates anxiety-like and social behaviors and regulates neuronal excitability in the locus coeruleus.

RTL1/PEG11, which has been associated with anxiety disorders, is a retrotransposon-derived imprinted gene in the placenta. However, imprinting patterns and functions of RTL1 in the brain have not been well-investigated. We found Rtl1 was paternally, but not maternally, expressed in brain stem, thalamus, and hypothalamus of mice, and imprinting status of RTL1 was maintained in human brain. Paternal Rtl1 knockout (Rtl1m+/p-) mice had higher neonatal death rates due to impaired suckling, and low body weights beginning on embryonic day 16.5. High paternal expression of Rtl1 was detected in the locus coeruleus (LC) and Rtl1m+/p- mice showed an increased delay in time of onset for action potentials and inward currents with decreased neuronal excitability of LC neurons. Importantly, Rtl1m+/p- mice exhibited behaviors associated with anxiety, depression, fear-related learning and memory, social dominance, and low locomotor activity. Taken together, our findings demonstrate RTL1 is imprinted in brain, mediates emotional and social behaviors, and regulates excitability in LC neurons.

Lactate peak in muscle disclosed by magnetic resonance spectroscopy in a patient with CPEO-plus syndrome.

A 25-year-old man complained of progressive diplopia and limb weakness for 3 years. Mitochondrial myopathy was suspected according to clinical presentation, elevated serum lactate concentration, and muscle histopathology. However, next-generation mtDNA sequencing (mtDNA NGS) of the blood only revealed a likely benign variant in the MT-CO1 gene (m.6510G > A). An mtDNA NGS study on the muscle sample revealed a large mtDNA deletion (m.5788-m.16071). The patient was diagnosed as having CPEO-plus syndrome related to the large mtDNA deletion. Notably, magnetic resonance spectroscopy revealed a doublet peak at 1-2 ppm in his edematous right vastus lateralis, which indicated lactate accumulation. Thus, muscle imaging and appropriate genetic tests facilitated the diagnosis of mitochondrial myopathy.

A systematic review of late-onset and very-late-onset multiple acyl-coenzyme A dehydrogenase deficiency: Cohort analysis and patient report from Taiwan.

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is a rare metabolic disorder with a dramatic clinical presentation. It was recently discovered that MADD may present at an advanced age. The clinical and laboratory data of an index patient and patients previously diagnosed at our institution were collected. A systematic review of previous studies retrieved from the PubMed, MEDLINE, and Embase databases published by February 1, 2020 was performed to collect patients with very-late-onset MADD (VLO-MADD, onset age > 60 years) globally and patients with late-onset MADD (LO-MADD, onset age < 60 years) in Taiwan. The clinical characteristics of the VLO-MADD patients were compared to those of LO-MADD patients. We report a patient with VLO-MADD who developed the first symptom at the age of 61 years. The patient presented with a Reye-like syndrome after taking aspirin for coronary artery disease. Repeated bouts of weakness were noted. Two variants of c.250 G > A (;) 419C > T were observed in the ETFDH gene. Another four patients with VLO-MADD were identified globally. Eighteen patients with LO-MADD were collected from our department and previously reported patients in Taiwan. There was no difference in the clinical symptoms (except for the onset age) or laboratory data between these two groups. Homozygous variants were not observed in any patients in the VLO-MADD group but were detected in 12 patients (66.6%) in the LO-MADD group (p = 0.014). Patients with MADD may first show symptoms in their 6th decade or beyond. The disease course may lead to erroneous diagnoses in this age group.

Clinical features of Pompe disease with motor neuronopathy.

Pathological studies on rodent models and patients with Pompe disease have demonstrated the accumulation of glycogen in spinal motor neurons; however, this finding has rarely been evaluated clinically in patients with Pompe disease. In this study, we analyzed seven patients (age, 7-11 years) with Pompe disease who received long-term enzyme replacement therapy. In addition to traditional myopathy-related clinical and electrophysiological features, these patients often developed bilateral foot drop, distal predominant weakness of four limbs, and hypo- or areflexia with preserved sensory function. Electrophysiological studies showed not only reduced amplitudes of compound muscle action potential, but also absent or impersistent F waves and mixed small and large/giant polyphasic motor unit action potentials with normal sensory study. Muscle biopsy usually showed the existence of angular fingers, fiber type grouping or group atrophy. Taken together, these features support the co-existence of motor neuronopathy additionally to myopathy.

Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of Pkd1 to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an in vitro cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Nuclear immunoreactivity of BLM-s, a proapoptotic BCL-2 family member, is specifically detected in salivary adenoid cystic carcinoma.

Tumor cells frequently evade apoptosis triggered by cellular stress via aberrant regulation of the BCL-2 family members, which are key players in regulating cell death under physiological and pathological situations. Previously, we have identified a novel BH3-only protein of the BCL-2 family, BLM-s (BCL-2-like molecule, short form), that modulates apoptosis of postmitotic immature neurons during corticohistogenesis. Whether BLM-s expression correlates with any subtype of human tumors has not been investigated. Here, via BLM-s immunohistochemistry performed in various kinds of human tumors, we demonstrate that BLM-s is specifically expressed in tumors derived from salivary gland (specificity, 0.76 [95% confidence interval, or CI], 0.65-0.85]; sensitivity, 1 [95% CI, 0.99-1]). Stratification of BLM-s immunointensity and its subcellular localization in correlation with salivary gland tumor subtype shows a statistically significant increase in proportion and in intensity of nuclear staining for adenoid cystic carcinoma (ACC; specificity, 0.92 [95% CI, 0.88-0.95]; sensitivity, 0.82 [95% CI, 0.66-0.92]), a locally aggressive head and neck malignancy. Comparison among salivary ACC in correlation with MYB/MYBL fluorescence in situ hybridization, c-KIT immunohistochemistry, and BLM-s immunohistochemistry shows that BLM-s' nuclear immunoreactivity has lower false-negative detection rate (18.5% compared with 26.3% [MYB/MYBL fluorescence in situ hybridization] and 34.2% [c-KIT], respectively). Intriguingly, ACC derived from other cell origins such as breast shows negative BLM-s immunoreactivity. We thus propose that nuclear localization of BLM-s detected by immunohistochemistry could be potentially used as an ancillary diagnostic marker for ACC originating from the salivary gland, especially when the biopsy specimen is small with an unknown tumor origin.

Regulation of axon repulsion by MAX-1 SUMOylation and AP-3.

During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In Caenorhabditis elegans, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5-mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5-mediated axon repulsion in C. elegans motor axons requires both max-1 SUMOylation and the AP-3 complex ß subunit gene, apb-3 Genetic interaction studies show that max-1 is SUMOylated by gei-17/PIAS1 and acts upstream of apb-3 Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3-mediated trafficking and degradation of UNC-5 receptors during axon guidance.

Wild-type p53 upregulates an early onset breast cancer-associated gene GAS7 to suppress metastasis via GAS7-CYFIP1-mediated signaling pathway.

The early onset breast cancer patients (age ≤ 40) often display higher incidence of axillary lymph node metastasis, and poorer five-year survival than the late-onset patients. To identify the genes and molecules associated with poor prognosis of early onset breast cancer, we examined gene expression profiles from paired breast normal/tumor tissues, and coupled with Gene Ontology and public data base analysis. Our data showed that the expression of GAS7b gene was lower in the early onset breast cancer patients as compared to the elder patients. We found that GAS7 was associated with CYFIP1 and WAVE2 complex to suppress breast cancer metastasis via blocking CYFIP1 and Rac1 protein interaction, actin polymerization, and ß1-integrin/FAK/Src signaling. We further demonstrated that p53 directly regulated GAS7 gene expression, which was inversely correlated with p53 mutations in breast cancer specimens. Our study uncover a novel regulatory mechanism of p53 in early onset breast cancer progression through GAS7-CYFIP1-mediated signaling pathways.

Allele-specific expression in a family quartet with autism reveals mono-to-biallelic switch and novel transcriptional processes of autism susceptibility genes.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, and the exact causal mechanism is unknown. Dysregulated allele-specific expression (ASE) has been identified in persons with ASD; however, a comprehensive analysis of ASE has not been conducted in a family quartet with ASD. To fill this gap, we analyzed ASE using genomic DNA from parent and offspring and RNA from offspring's postmortem prefrontal cortex (PFC); one of the two offspring had been diagnosed with ASD. DNA- and RNA-sequencing revealed distinct ASE patterns from the PFC of both offspring. However, only the PFC of the offspring with ASD exhibited a mono-to-biallelic switch for LRP2BP and ZNF407. We also identified a novel site of RNA-editing in KMT2C in addition to new monoallelically-expressed genes and miRNAs. Our results demonstrate the prevalence of ASE in human PFC and ASE abnormalities in the PFC of a person with ASD. Taken together, these findings may provide mechanistic insights into the pathogenesis of ASD.

Blm-s, a BH3-only protein enriched in postmitotic immature neurons, is transcriptionally upregulated by p53 during DNA damage.

Programmed cell death is a pivotal process that regulates neuronal number during development. Key regulators of this process are members of the BCL-2 family. Using mRNA differential display, we identified a Bcl-2 family gene, Blm-s (Bcl-2-like molecule, short form), enriched in postmitotic neurons of the developing cerebral cortex. BLM-s functions as a BH3-only apoptosis sensitizer/derepressor and causes BAX-dependent mitochondria-mediated apoptosis by selectively binding to prosurvival BCL-2 or MCL-1. When challenged with γ-irradiation that produces DNA double-strand breaks (DSBs), Blm-s is transcriptionally upregulated in postmitotic immature neurons with concurrently increased apoptosis. RNAi-mediated depletion of Blm-s protects immature neurons from irradiation-induced apoptosis. Furthermore, Blm-s is a direct target gene of p53 and AP1 via the ataxia telangiectasia mutated (ATM)- and c-Jun N-terminal kinase (JNK)-signaling pathways activated by DSBs. Thus, BLM-s is likely an apoptosis sensor activated by DSBs accumulating in postmitotic immature neurons.

Peripancreatic schwannoma.

BACKGROUND: The extrapancreatic nerve plexus may give rise to schwannomas. The aim of this study was to define the clinicopathologic features of peripancreatic schwannoma and to allow improved diagnosis and treatment for this condition. METHODS: A retrospective analysis was performed on 17 patients with pathologically proven peripancreatic schwannoma (defined as schwannoma in anatomic proximity to the pancreas) treated at our hospital between 1995 and 2011. RESULTS: The patient group included 9 men and 8 women between the ages of 26 and 67 years. The tumor size ranged from 2.5 to 13 cm. Tumor locations were the pancreatic head plexus in 5 patients, the superior mesenteric plexus in 3, the pancreatic head and superior mesenteric plexus in 2, the celiac plexus in 4, and the splenic plexus in 3. The main symptoms were vague abdominal pain (n = 5; 29.4%) and abdominal mass (n = 2; 11.8%). A correct preoperative diagnosis was made in 12 patients by either computed tomography (CT) or magnetic resonance imaging (MRI). Sixteen patients (94.1%) underwent total resection and 1 patient (5.9%) underwent subtotal resection. Three patients underwent concomitant pancreatectomy. No recurrences were noted in the 16 patients who underwent total tumor resection. CONCLUSION: Most peripancreatic schwannomas are benign. Peripancreatic schwannoma may often be accurately diagnosed before surgery by its typical location and CT or MRI findings. Total resection is important for treatment, and no recurrences are seen when resection is complete. Prognosis after total resection is extremely good.

Resolution of diabetes after pancreaticoduodenectomy in patients with and without pancreatic ductal cell adenocarcinoma.

BACKGROUND: New-onset diabetes mellitus (DM) is associated with pancreatic ductal cell adenocarcinoma (PDCA) and can resolve after pancreaticoduodenectomy (PD). Whether DM also resolves after PD in patients operated for disease other than PDCA remains to be determined. METHODS: We compared glycemic status before and after PD between patients with and without PDCA by review of a prospectively maintained database including all patients receiving PD from 2005 to 2011. New-onset DM was defined as diagnosis of DM less than 24 months before PD, and cases with DM diagnosis≥24 months preceding PD were described as long-standing DM. RESULTS: Of 458 patients receiving PD, there were 146 (31.9%) PDCA and 312 (68.1%) non-PDCA, including 160 benign diseases, 113 ampulla cancer, 29 distal common bile duct cancer, and 10 duodenal cancer. Overall prevalence of DM was higher in PDCA group than non-PDCA group (37.7 vs. 25.6%; P=0.011). Resolution of new-onset DM after PD was observed in 9 (41%) of 22 patients with PDCA and in 12 (63%) of 19 patients without PDCA. Resolution of long-standing DM after PD was also observed in 3 (9.1%) of 33 patients with PDCA and in 6 (9.8%) of 61 patients without PDCA. CONCLUSIONS: DM resolved after PD in some patients both with and without PDCA. These findings suggest that PD-associated anatomic change may play a role in resolution of DM after PD.