Lengthening a glutamine tract in huntingtin confers a dominant attribute that initiates degeneration of striatal neurons in Huntington’s disease (HD). To identify pathways that are candidates for the mutant protein’s abnormal function, we compared striatal cell lines established from wild-type and Hdh^Q111 knock-in embryos. Alternate versions of full-length huntingtin, distinguished by epitope accessibility, were localized to different sets of nuclear and perinuclear organelles involved in RNA biogenesis and membrane trafficking. However, mutant STHdh^Q111 cells also exhibited additional forms of the full-length mutant protein and displayed dominant phenotypes that did not mirror phenotypes caused by either huntingtin deficiency or excess. These phenotypes indicate a disruption of striatal cell homeostasis by the mutant protein, via a mechanism that is separate from its normal activity. They also support specific stress pathways, including elevated p53, endoplasmic reticulum stress response and hypoxia, as potential players in HD.

Point mutations of the CACNA1A gene coding for the α_1A voltage-dependent calcium channel subunit are responsible for familial hemiplegic migraine (FHM) and episodic ataxia type 2 (EA2). In addition, expansions of the CAG repeat motif at the 3′ end of the gene, smaller than those responsible for dynamic mutation disorders, were found in patients with a progressive spinocere-bellar ataxia, named SCA6. In the present work, the analysis of two new families with small CAG expansions of the CACNA1A gene is presented. In one family, with a clinical diagnosis of EA2, a CAG₂₃ repeat allele segregated in patients showing different interictal symptoms, ranging from nystagmus only to severe progressive cerebellar ataxia. No additional mutations in coding and intron-exon junction sequences in disequilibrium with the CAG expansion were found. In the second family, initially classified as autosomal dominant cerebellar ataxia of unknown type, an inter-generational allele size change showed that a CAG₂₀ allele was associated with an EA2 phenotype and a CAG₂₅ allele with progressive cerebellar ataxia. These results show that EA2 and SCA6 are the same disorder with a high pheno-typic variability, at least partly related to the number of repeats, and suggest that the small expansions may not be as stable as previously reported. A refinement of the coding and intron-exon junction sequences of the CACNA1A gene is also provided.

Huntington’s disease (HD), with its selective neuronal cell loss, is caused by an elongated glutamine tract in the huntingtin protein. To discover the pathways that are candidates for the protein’s normal and/or abnormal function, we surveyed 19 classes of organelle in Hdh^ex4/5/Hdh^ex4/5 knock-out compared with wild-type embryonic stem cells to identify any that might be affected by huntingtin deficiency. Although the majority did not differ, dramatic changes in six classes revealed that huntingtin’s function is essential for the normal nuclear (nucleoli, transcription factor-speckles) and perinuclear membrane (mitochondria, endoplasmic reticulum, Golgi and recycling endosomes) organelles and for proper regulation of the iron pathway. Moreover, upmodulation by deferoxamine mesylate implicates huntingtin as an iron-response protein. However, excess huntingtin produced abnormal organelles that resemble the deficiency phenotype, suggesting the importance of huntingtin level to the protein’s normal pathway. Thus, organelles that require huntingtin to function suggest roles for the protein in RNA biogenesis, trafficking and iron homeostasis to be explored in HD pathogenesis.

Prolonged culturing of rodent cells in vitro activates p19^ARF (named p14^ARF in man), resulting in a p53-dependent proliferation arrest known as senescence. The p19^ARF-Mdm2-p53 pathway also serves to protect primary cells against oncogenic transformation. We have used a genetic screen in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen, to identify genes that allow bypass of senescence. Using retroviral cDNA expression libraries, we have identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-box gene, which is found mutated in the human developmental disorder Ulnar-Mammary Syndrome. We have shown that TBX-3 potently represses expression of both mouse p19^ARF and human p14^ARF. We have also shown here that point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome, have lost the ability to inhibit senescence and fail to repress mouse p19^ARF and human p14^ARF expression. These data suggest that the hypoproliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14^ARF.

We examined the effects of the chemokine fractalkine (CX₃CL1) on EPSCs evoked by electrical stimulation of Schaffer collaterals in patch-clamped CA1 pyramidal neurons from rat hippocampal slices. Acute application of CX₃CL1 caused a sustained reduction of EPSC amplitude, with partial recovery after washout. CX₃CL1-induced EPSC depression is postsynaptic in nature, because paired-pulse ratio was maintained, amplitude distribution of spontaneous excitatory postsynaptic currents shifted to lower values, and whole-cell current responses to AMPA were reversibly inhibited. EPSC depression by CX₃CL1 is mediated by CX₃CL1 receptor (CX₃CR1), because CX₃CL1 was unable to influence EPSC amplitude in CA1 pyramidal neurons from CX₃CR1 knock-out mice. CX₃CL1-induced depression of both EPSC and AMPA current was not observed in the absence of afferent fiber stimulation or AMPA receptor activation, respectively, indicating the requirement of sustained receptor activity for its development. Findings obtained from hippocampal slices, cultured hippocampal neurons, and transfected human embryonic kidney cells indicate that a Ca²⁺-, cAMP-, and phosphatase-dependent process is likely to modulate CX₃CL1 effects because of the following: (1) CX₃CL1-induced depression was antagonized by intracellular BAPTA, 8Br-cAMP, phosphatase inhibitors, and pertussis toxin (PTX); (2) CX₃CL1 inhibited forskolin-induced cAMP formation sensitive to PTX; and (3) CX₃CL1 inhibited forskolin-induced Ser845 GluR1 phosphorylation, which was sensitive to PTX and dependent on Ca²⁺and phosphatase activity. Together, these findings indicate that CX₃CL1 negatively modulates AMPA receptor function at active glutamatergic synapses through cell-signaling pathways by influencing the balance between kinase and phosphatase activity.