I would also like to thank my teachers who inspired my initial interest in biomedical science research. There are also many friends I would like to thank for their contribution to my experience at this graduate school. Finally, I would like to thank my parents in China, my mother and my father.
The family of cation-chloride cotransporters
The phenotypes of these two kidney diseases include an imbalance of ions resulting in hypokalemic metabolic alkalosis, hypomagnesemia, and hypercalciuria (Roser et al., 2009). In this situation, when GABA binds to its receptor, the current becomes hyperpolarizing and inhibitory, which is the normal state for mature neurons (Ben-Ari et al., 2012; Lu et al., 1999; Plotkin et al., 1997 ). (Figure 2). The high expression and activity of NKCC1 leads to accumulation of [Cl−]i, leading to partial depolarization of the terminals of primary afferent neurons.
The K-Cl cotransporter-3, KCC3
Discovery and Structure
Due to the high activity of NKCC1, intracellular chloride. concentration is high and causes depolarizing GABAergic currents. Our laboratory showed that SPAK and OSR1 are two kinases that directly regulate KCC activity (Piechotta et al., 2002). There are several reports showing that WNK kinases and SPAK and OSR1 kinases in heterologous systems inhibit K-Cl cotransport, while another study failed to observe an inhibitory effect of SPAK on KCC through.
Spatial KCC3 Expression
KCC3 function and activity
Transported ions, tissue distribution in mammals and related disease due to genetic mutations in humans are shown. For KCC proteins, there are 12 transmembrane domains, a large extracellular loop between the fifth and sixth transmembrane domains, and a long carboxyl terminus.
Peripheral Neuropathy Disorders
Another common neuropathy is idiopathic neuropathy, which means that the cause of this neuropathy is unknown (Hughes, 1995). In other parts of the world such as Southeast Asia, India, Africa, the highly prevalent disease of leprosy neuritis is still the leading cause of neuropathy (Martyn and Hughes, 1997). There is currently no effective and powerful strategy to completely cure this disorder, most current treatment methods focus on improving the patient's conditions (Torpy et al., 2010).
HMSN/ACC
Disease overview
It is a very rare hereditary human disease with only a few sporadic reports available from most parts of the world. Since its first description, extensive studies have been carried out to uncover the cause of the disease. In fact, most of the known ACCPN mutations result in a truncated KCC3 protein where the C-terminus is absent, suggesting a possible role of the C-terminus of KCC3 in the.
Animal models
Further analysis of the sciatic nerves of the mutant mice revealed axonal and periaxonal swelling and axon and myelin. The electrocorticograms of the mutant mice showed a very similar pattern to those obtained in patients with. As mentioned above, the arterial blood pressure of the mutant mice was significantly higher than that of control mice, likely due to increased sympathetic tone.
Creation of genetically-modified mouse models
To further define the role of KCC3 in the pathogenic mechanisms leading to ACCPN, Guy Rouleau and colleagues recently generated a neuron-specific KCC3-null mouse line (Shekarabi et al., 2012). This can be achieved either by inserting a piece of DNA or by removing an exon in order to disrupt the transcript (Anagnostopoulos et al., 2001). To obtain the expression of an altered protein, a knock-in mutation can be introduced into the mouse genome, using the same technology for knock-outs (Roebroek et al., 2011).
Expression and trafficking of the KCC3-E289G protein will be investigated in both heterologous expression systems. This will be achieved by knocking in mutated exon 7 by homologous recombination, obtaining viable homozygous animals and studying its locomotor phenotype.
Characterization of KCC3 E289G mutant in vitro
- Introduction
- Methods
- Results
- Discussion
We demonstrate that the KCC3-E289G mutant protein resides in the endoplasmic reticulum (ER), is not properly glycosylated, and does not traffic to the plasma membrane. In Figure 8, we co-expressed wild-type KCC3 and KCC3-E289G in Xenopus laevis oocytes and used co-immunoprecipitation to show specific proteins. Because KCC3 is inactive under isosmotic conditions, we also examined the effect of wild-type KCC3 and KCC3-E289G on KCC2 under hypotonic conditions.
Xenopus laevis oocytes were injected with KCC2 cRNA in the presence or absence of wild-type KCC3 or KCC3-E289G cRNA. Xenopus laevis oocytes were injected with KCC2 in the presence or absence of wild-type KCC3 or KCC3-E289G cRNA, and membrane fractions were isolated using the silica cross-linking method. The KCC3-E289G mutant prevented wild-type KCC3 from reaching the plasma membrane (Figure 10A), resulting in a dominant negative effect on KCC3 function (Figure 10B).
The lower molecular size of the KCC3-E289G band observed in Figure 10 indicates a defect in glycosylation. To further substantiate this defect, we transfected HEK 293FT cells with wild-type and mutant KCC3 and observed the absence of larger molecular weight products in the KCC3-E289G mutant (Figure 12A). Because KCC3 is naturally expressed in Chinese hamster ovary (CHO) cells, we used mutant CHO cell lines (Figure 12) to evaluate the extent of the deficiency in KCC3-E289G glycosylation.
Coimmunoprecipitation experiments revealed that both wild-type and the KCC3-E289G mutant interact with KCC2 when co-injected into oocytes. Western blot analysis data show the absence of a broad band characteristic of a glycosylated membrane protein for the KCC3-E289G mutant. Experiments performed in Xenopus laevis oocytes and mammalian HEK 293FT cells revealed that the KCC3-E289G mutant cotransporter is stuck in the endoplasmic reticulum and likely receives only core mannose glycosylation.
Characterization of KCC3-E289G mutant in vivo
Briefly, a 2526 bp fragment (short arm) from the BAC clone was dropped between unique sites upstream of the first loxP site. Finally, a larger fragment of 7.5 kb (large arm) was removed from the BAC clone downstream of the last loxP site. The mice were placed in the center of the open field and their activity was monitored for 60 minutes.
Distance traveled and time spent in the periphery (thigmotaxis) of the chamber were determined by the number of beam crossings. DRGs were dissected from the lower thoracic to mid-lumbar regions of the vertebral column and placed in 0.1 M PBS. Dorsal root ganglia were dissected from the lower thoracic to mid-lumbar regions of the vertebral column and placed in a 4%.
To generate the KCC3-E289G knockout in mice, we generated a targeting vector that included the E289G mutation within exon 7 of the Slc12a6 gene. After germline transfer, elimination of the neomycin resistance gene cassette by crossing mutant mice with FlpE mice, the 3 kb fragment was amplified by PCR to demonstrate recombination at the appropriate locus. Most other KCC3 mutations affect plasma membrane localization of the mutant transporter, preventing normal trafficking to the membrane with significant retention of the protein in the endoplasmic reticulum (ER).
If some of the KCC3 mutant proteins were successfully trafficked, they could function at the plasma membrane and thereby rescue the locomotor phenotype.
Cellular origin of ACCPN: Tissue-specific knockouts
Deletion of KCC3 in desert hedgehog driven Schwann cells also failed to induce a locomotor phenotype. To confirm that neurons were involved in the development of the phenotype, we used enolase-2-CRE (Eno2-CRE) mice to induce KCC3 deletion in neurons. Large vacuoles reminiscent of those seen in the brain and spinal cord with global KCC3 knockout (Boettger et al., 2003) were observed in the dorsal roots of Prvlb-CRE x KCC3 knockout mice (Figure 28, arrows).
In addition, many structures containing dense material (arrowheads) were also observed in the DRG of these mice. The locomotor deficit observed in the Prvlb-CRE x KCC3f/f mice is unlikely to be related to a disruption of parvalbumin in these mice. Furthermore, disruption of parvalbumin in mice was shown to have no phenotype in the rotarod assay (Farré-Castany et al., 2007).
In fact, we demonstrated that the abnormal structures in dorsal root ganglia are tissue-specific or global knockouts of KCC3, ie. this observation makes the involvement of CNS interneurons (eg in the cerebellum) less likely in the locomotor phenotype. Dhh is specifically expressed in Schwann cells of the nervous system and Sertoli cells in the mammalian testis.
Our results showed no locomotor phenotype in these mice, indicating no participation of Schwann cell KCC3 in the development of ACCPN. Since no locomotor deficit was observed in these mice, we can conclude that the accelerated rotarod phenotype observed in the global KCC3 knockout mice is not related to a role of the co-transporter in these small sensory fibers. In conclusion, our study establishes a key role for parvalbumin-expressing neurons in the development of the locomotor phenotype associated with HSN/ACC.
Conclusions and Future Directions
- Summary of work
- Future directions
In a separate study, I addressed the origin of the cells responsible for the development of ACCPN. Andermann F, Andermann E, Joubert M, Karpati G, Carpenter S, Melancon D (1972) Familial age of the corpus callosum with anterior horn cell disease. Andermann F, Andermann E, Joubert M, Karpati G, Carpenter S, Melancon D (1972) Familial age of the corpus callosum with anterior horn cell disease: a syndrome of mental retardation, areflexia and paraparesis.
De Braekeleer M, Dallaire A, Mathieu J (1993) Genetic epidemiology of sensorimotor polyneuropathy with or without agenesis of the corpus callosum in northeastern Quebec. Dupre N, Howard HC, Mathieu J, Karpati G, Vanasse M, Bouchard JP, Carpenter S, Rouleau GA (2003) Hereditary motor and sensory neuropathy with agenesis of the corpus callosum. Hauser E, Bittner R, Liegl C, Bernert G, Zeitlhofer J (1993) Incidence of Andermann syndrome out of French Canada - genesis of corpus callosum with neuronopathy.
Kahle KT, Rinehart J, Lifton RP (2010) Phosphoregulation of Na-K-2Cl and K-Cl cotransporters by WNK kinases. Labrisseau A, Vanasse M, Brochu P, Jasmin G (1984) Andermann syndrome: agenesis of the corpus callosum associated with mental retardation and progressive sesorimotor neuronopathy. Rathmayer W, Djokaj S (2000) Presynaptic inhibition and GABA(B) receptor involvement at neuromuscular junctions of the crab Eriphia spinifrons.
Shekarabi M, Salin-Cantegrel A, Laganiere J, Gaudet R, Dion P, Rouleau GA (2011) Cellular expression of the K+-Cl cotransporter KCC3 in the mouse central nervous system.
