Loss of Flot2 expression in deep cerebellar nuclei neurons of mice with Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is caused by a deficiency of the NPC1 or NPC2 gene, leading to storages of unesterified cholesterol and sphingolipids. Cerebellar ataxia is a main symptom of NPC and the deep cerebellar nuclei (DCN) is the sole signal output of the cerebellum. In this study, we explored the pathological changes in DCN neurons of Npc1 knockout mice (Npc1-). We first demonstrated that DCN neurons of Npc1- mice had prominent ganglioside GM2 accumulation in the late endosomes but not in the lysosomes. More importantly, Flot2 expression, a marker for the lipid rafts, was lost. Single-nucleus RNA sequencing analysis revealed a generalized reduction in gene expression in DCN neurons, though Camk1d, encoding one of the Ca2+/calmodulin-dependent protein kinases (CaMKs), increased in expression. We treated Npc1- mice with CaMK inhibitor KN-93, but CaMK1D expression increased further. We also fed Npc1- mice with two medications for NPC. We found that miglustat, a sphingolipid synthesis inhibitor, increased the expression of Flot2. Moreover, N-acetyl l-leucine (NALL), an experimental medicine for NPC, recovered Flot2 expression. Therefore, our data suggest that in Npc1- mice, GM2 sequestration and the loss of lipid rafts lead to cell dysfunction and symptoms of NPC.

Ultrastructural and diffusion tensor imaging studies reveal axon abnormalities in Pompe disease mice.

Pompe disease (PD) is caused by lysosomal glycogen accumulation in tissues, including muscles and the central nervous system (CNS). The intravenous infusion of recombinant human acid alpha-glucosidase (rhGAA) rescues the muscle pathologies in PD but does not treat the CNS because rhGAA does not cross the blood-brain barrier (BBB). To understand the CNS pathologies in PD, control and PD mice were followed and analyzed at 9 and 18 months with brain structural and ultrastructural studies. T2-weighted brain magnetic resonance imaging studies revealed the progressive dilatation of the lateral ventricles and thinning of the corpus callosum in PD mice. Electron microscopy (EM) studies at the genu of the corpus callosum revealed glycogen accumulation, an increase in nerve fiber size variation, a decrease in the g-ratio (axon diameter/total fiber diameter), and myelin sheath decompaction. The morphology of oligodendrocytes was normal. Diffusion tensor imaging (DTI) studies at the corpus callosum revealed an increase in axial diffusivity (AD) and mean diffusivity (MD) more significantly in 9-month-old PD mice. The current study suggests that axon degeneration and axon loss occur in aged PD mice and are probably caused by glycogen accumulation in neurons. A drug crossing the BBB or a treatment for directly targeting the brain might be necessary in PD.

Insight into a single halobacterium using a dual-bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination.

The light-driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource-limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual-BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo-BR). The contribution of this dual-BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH-dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual-HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light-driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.

A transducer for microbial sensory rhodopsin that adopts GTG as a start codon is identified in Haloarcula marismortui.

Microbial sensory rhodopsins are known to mediate phototaxis, and all of the known sensory rhodopsins execute this function with a specific cognate transducer that has two-transmembrane (2-TM) regions. In the genome of Haloarcula marismortui, a total of six rhodopsin genes were annotated, and we previously showed three of them to be the ion type and suggested the other three as sensory type, even though the candidate transducer gene, htr, for HmSRI was missing the 2-TM region that is found in all of the other known transducers. Here we showed this htr gene featured a preceding 2-TM region when the alternative start codon GTG located 291 nucleotides upstream of the original annotated open reading frame (ORF) was introduced and it is named as htrI in this study. Overexpression of HmHtrI exhibited it existed as a membrane protein and several biophysical assays confirmed it functionally interacted with HmSRI. Together with our previous reverse-transcriptase-PCR results and phototaxis measurements, the new ORF of original predicted soluble htr gene product was a membrane protein with a 2-TM region, HmHtrI; and it serves as the cognate transducer for HmSRI. HmHtrI therefore is the first transducer for the sensory rhodopsin adopted start codon other than ATG.