Mitochondria dysfunction occurs in the aging brain as well as in several neurodegenerative disorders and predisposes neuronal cells to enhanced sensitivity to neurotoxins. 3-nitropropionic acid (3-NP) is a naturally occurring plant and fungal neurotoxin that causes neurodegeneration predominantly in the striatum by irreversibly inhibiting the tricarboxylic acid respiratory chain enzyme, succinate dehydrogenase (SDH), the main constituent of the mitochondria respiratory chain complex II. Significantly, although 3-NP-induced inhibition of SDH occurs in all brain regions, neurodegeneration occurs primarily and almost exclusively in the striatum for reasons still not understood. In rodents, 3-NP-induced striatal neurodegeneration depends on the strain background suggesting that genetic differences among genotypes modulate toxicant variability and mechanisms that underlie 3-NP-induced neuronal cell death. Using the large BXD family of recombinant inbred (RI) strains we demonstrate that variants in Ccnd1 - the gene encoding cyclin D1 - of the DBA/2 J parent underlie the resistance to 3-NP-induced striatal neurodegeneration. In contrast, the Ccnd1 variant inherited from the widely used C57BL/6 J parental strain confers sensitivity. Given that cellular stress triggers induction of cyclin D1 expression followed by cell-cycle re-entry and consequent neuronal cell death, we sought to determine if the C57BL/6 J and DBA/2 J Ccnd1 variants are differentially modulated in response to 3-NP. We confirm that 3-NP induces cyclin D1 expression in striatal neuronal cells of C57BL/6 J, but this response is blunted in the DBA/2 J. We further show that striatal-specific alternative processing of a highly conserved 3'UTR negative regulatory region of Ccnd1 co-segregates with the C57BL/6 J parental Ccnd1 allele in BXD strains and that its differential processing accounts for sensitivity or resistance to 3-NP. Our results indicate that naturally occurring Ccnd1 variants may play a role in the variability observed in neurodegenerative disorders involving mitochondria complex II dysfunction and point to cyclin D1 as a possible therapeutic target.

The splice site mutation in the IKBKAP gene coding for IKAP protein leads to the tissue-specific skipping of exon 20, with concomitant reduction in IKAP protein production. This causes the neurodevelopmental, autosomal-recessive genetic disorder - Familial Dysautonomia (FD). The molecular hallmark of FD is the severe reduction of IKAP protein in the nervous system that is believed to be the main reason for the devastating symptoms of this disease. Our recent studies showed that in the brain of two FD patients, genes linked to oligodendrocyte differentiation and/or myelin formation are significantly downregulated, implicating IKAP in the process of myelination. However, due to the scarcity of FD patient tissues, these results awaited further validation in other models. Recently, two FD mouse models that faithfully recapitulate FD were generated, with two types of mutations resulting in severely low levels of IKAP expression. Here we demonstrate that IKAP deficiency in these FD mouse models affects a similar set of genes as in FD patients' brains. In addition, we identified two new IKAP target genes involved in oligodendrocyte cells differentiation and myelination, further underscoring the essential role of IKAP in this process. We also provide proof that IKAP expression is needed cell-autonomously for the regulation of expression of genes involved in myelin formation since knockdown of IKAP in the Oli-neu oligodendrocyte precursor cell line results in similar deficiencies. Further analyses of these two experimental models will compensate for the lack of human postmortem tissues and will advance our understanding of the role of IKAP in myelination and the disease pathology.

Loss-of-function mutations in SGCE, which encodes ε-sarcoglycan (ε-SG), cause myoclonus-dystonia syndrome (OMIM159900, DYT11). A "major" ε-SG protein derived from CCDS5637.1 (NM_003919.2) and a "brain-specific" protein, that includes sequence derived from alternative exon 11b (CCDS47642.1, NM_001099400.1), are reportedly localized in post- and pre-synaptic membrane fractions, respectively. Moreover, deficiency of the "brain-specific" isoform and other isoforms derived from exon 11b may be central to the pathogenesis of DYT11. However, no animal model supports this hypothesis. Gene-trapped ES cells (CMHD-GT_148G1-3, intron 9 of NM_011360) were used to generate a novel Sgce mouse model (C57BL/6J background) with markedly reduced expression of isoforms derived from exons 3' to exon 9 of NM_011360. Among those brain regions analyzed in adult (2month-old) wild-type (WT) mice, cerebellum showed the highest relative expression of isoforms incorporating exon 11b. Homozygotes (SgceGt(148G1)Cmhd/Gt(148G1)Cmhd or SgceGt/Gt) and paternal heterozygotes (Sgcem+/pGt, m-maternal, p-paternal) showed 60 to 70% reductions in expression of total Sgce. Although expression of the major (NM_011360) and brain-specific (NM_001130189) isoforms was markedly reduced, expression of short isoforms was preserved and relatively small amounts of chimeric ε-SG/β-galactosidase fusion protein was produced by the Sgce gene-trap locus. Immunoaffinity purification followed by mass spectrometry assessments of Sgcem+/pGt mouse brain using pan- or brain-specific ε-SG antibodies revealed significant reductions of ε-SG and other interacting sarcoglycans. Genome-wide gene-expression data using RNA derived from adult Sgcem+/pGt mouse cerebellum showed that the top up-regulated genes were involved in cell cycle, cellular development, cell death and survival, while the top down-regulated genes were associated with protein synthesis, cellular development, and cell death and survival. In comparison to WT littermates, Sgcem+/pGt mice exhibited "tiptoe" gait and stimulus-induced appendicular posturing between Postnatal Days 14 to 16. Abnormalities noted in older Sgcem+/pGt mice included reduced body weight, altered gait dynamics, and reduced open-field activity. Overt spontaneous or stimulus-sensitive myoclonus was not apparent on the C57BL/6J background or mixed C57BL/6J-BALB/c and C57BL/6J-129S2 backgrounds. Our data confirm that mouse Sgce is a maternally imprinted gene and suggests that short Sgce isoforms may compensate, in part, for deficiency of major and brain-specific Sgce isoforms.

Familial Dysautonomia (FD) is an autosomal recessive disorder that affects 1/3,600 live births in the Ashkenazi Jewish population, and leads to death before the age of 40. The disease is characterized by abnormal development and progressive degeneration of the sensory and autonomic nervous system. A single base pair substitution in intron 20 of the Ikbkap gene accounts for 98% of FD cases, and results in the expression of low levels of the full-length mRNA with simultaneous expression of an aberrantly spliced mRNA in which exon 20 is missing. To date, there is no animal model for the disease, and the essential cellular functions of IKAP--the protein encoded by Ikbkap--remain unknown. To better understand the normal function of IKAP and in an effort to generate a mouse model for FD, we have targeted the mouse Ikbkap gene by homologous recombination. We created two distinct alleles that result in either loss of Ikbkap expression, or expression of an mRNA lacking only exon 20. Homozygosity for either mutation leads to developmental delay, cardiovascular and brain malformations, accompanied with early embryonic lethality. Our analyses indicate that IKAP is essential for expression of specific genes involved in cardiac morphogenesis, and that cardiac failure is the likely cause of abnormal vascular development and embryonic lethality. Our results also indicate that deletion of exon 20 abolishes gene function. This implies that the truncated IKAP protein expressed in FD patients does not retain any significant biological function.

Huntington's disease (HD) is caused by an autosomal dominant polyglutamine expansion mutation of Huntingtin (HTT). HD patients suffer from progressive motor, cognitive, and psychiatric impairments, along with significant degeneration of the striatal projection neurons (SPNs) of the striatum. HD is widely accepted to be caused by a toxic gain-of-function of mutant HTT. However, whether loss of HTT function, because of dominant-negative effects of the mutant protein, plays a role in HD and whether HTT is required for SPN health and function are not known. Here, we delete Htt from specific subpopulations of SPNs using the Cre-Lox system and find that SPNs require HTT for motor regulation, synaptic development, cell health, and survival during aging. Our results suggest that loss of HTT function in SPNs could play a critical role in HD pathogenesis.

The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the population suffer nerve degeneration or peripheral damage. For example, over 40% of patients with diabetes or undergoing chemotherapy develop peripheral neuropathies. Despite this, there are major gaps in the knowledge of human PNS development and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder that specifically affects the PNS making it an ideal model to study PNS dysfunction. FD is caused by a homozygous point mutation in ELP1 leading to developmental and degenerative defects in the sensory and autonomic lineages. We previously employed human pluripotent stem cells (hPSCs) to show that peripheral sensory neurons (SNs) are not generated efficiently and degenerate over time in FD. Here, we conducted a chemical screen to identify compounds able to rescue this SN differentiation inefficiency. We identified that genipin, a compound prescribed in Traditional Chinese Medicine for neurodegenerative disorders, restores neural crest and SN development in FD, both in the hPSC model and in a FD mouse model. Additionally, genipin prevented FD neuronal degeneration, suggesting that it could be offered to patients suffering from PNS neurodegenerative disorders. We found that genipin crosslinks the extracellular matrix, increases the stiffness of the ECM, reorganizes the actin cytoskeleton, and promotes transcription of YAP-dependent genes. Finally, we show that genipin enhances axon regeneration in an in vitro axotomy model in healthy sensory and sympathetic neurons (part of the PNS) and in prefrontal cortical neurons (part of the central nervous system, CNS). Our results suggest genipin can be used as a promising drug candidate for treatment of neurodevelopmental and neurodegenerative diseases, and as a enhancer of neuronal regeneration.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays an essential role in cellular and systemic O(2) homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1alpha in the brain, we exposed adult mice with late-stage brain deletion of HIF-1alpha to hypoxic injuries. Contrary to expectations, the brains from the HIF-1alpha-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1alpha can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1alpha-deficient mice. Although the role of HIF-1alpha has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1alpha in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1alpha-deficient mice is mechanistically consistent with a predominant role of HIF-1alpha as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1alpha, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington's disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD.

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspects of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test the in vivo efficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model, TgFD9; IkbkapΔ20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correct ELP1 splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a splice mutation in Elongator complex protein 1 (ELP1, also known as IKBKAP); this mutation leads to variable skipping of exon 20 and to a drastic reduction of ELP1 in the nervous system. Clinically, many of the debilitating aspects of the disease are related to a progressive loss of proprioception; this loss leads to severe gait ataxia, spinal deformities, and respiratory insufficiency due to neuromuscular incoordination. There is currently no effective treatment for FD, and the disease is ultimately fatal. The development of a drug that targets the underlying molecular defect provides hope that the drastic peripheral neurodegeneration characteristic of FD can be halted. We demonstrate herein that the FD mouse TgFD9;IkbkapΔ20/flox recapitulates the proprioceptive impairment observed in individuals with FD, and we provide the in vivo evidence that postnatal correction, promoted by the small molecule kinetin, of the mutant ELP1 splicing can rescue neurological phenotypes in FD. Daily administration of kinetin starting at birth improves sensory-motor coordination and prevents the onset of spinal abnormalities by stopping the loss of proprioceptive neurons. These phenotypic improvements correlate with increased amounts of full-length ELP1 mRNA and protein in multiple tissues, including in the peripheral nervous system (PNS). Our results show that postnatal correction of the underlying ELP1 splicing defect can rescue devastating disease phenotypes and is therefore a viable therapeutic approach for persons with FD.

Huntington's Disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by cognitive, behavioral and motor dysfunctions. HD is caused by a CAG repeat expansion in exon 1 of the HD gene that is translated into an expanded polyglutamine tract in the encoded protein, huntingtin (HTT). While the most significant neuropathology of HD occurs in the striatum, other brain regions are also affected and play an important role in HD pathology. To date there is no cure for HD, and recently strategies aiming at silencing HTT expression have been initiated as possible therapeutics for HD. However, the essential functions of HTT in the adult brain are currently unknown and hence the consequence of sustained suppression of HTT expression is unpredictable and can potentially be deleterious. Using the Cre-loxP system of recombination, we conditionally inactivated the mouse HD gene homologue at 3, 6 and 9 months of age. Here we show that elimination of Htt expression in the adult mouse results in behavioral deficits, progressive neuropathological changes including bilateral thalamic calcification, and altered brain iron homeostasis.

Despite being an autosomal dominant disorder caused by a known coding mutation in the gene HTT, Huntington's disease (HD) patients with similar trinucleotide repeat mutations can have an age of onset that varies by decades. One likely contributing factor is the genetic heterogeneity of patients that might modify their vulnerability to disease. We report that although the heterozygous depletion of the autophagy adaptor protein Alfy/Wdfy3 has no consequence in control mice, it significantly accelerates age of onset and progression of HD pathogenesis. Alfy is required in the adult brain for the autophagy-dependent clearance of proteinaceous deposits, and its depletion in mice and neurons derived from patient fibroblasts accelerates the aberrant accumulation of this pathological hallmark shared across adult-onset neurodegenerative diseases. These findings indicate that selectively compromising the ability to eliminate aggregated proteins is a pathogenic driver, and the selective elimination of aggregates may confer disease resistance.

Microtubule (MT)-based transport is an evolutionary conserved process finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, primarily catalyzed by a vesicular pool of α-tubulin N-acetyltransferase 1 (Atat1), promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanism that controls Atat1 activity remains poorly understood. Here, we show that ATP-citrate lyase (Acly) is enriched in vesicles and provide Acetyl-Coenzyme-A (Acetyl-CoA) to Atat1. In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in a pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine light on the pathophysiological mechanisms underlying FD.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a poly-glutamine (poly-Q) stretch in the huntingtin (Htt) protein. Gain-of-function effects of mutant Htt have been extensively investigated as the major driver of neurodegeneration in HD. However, loss-of-function effects of poly-Q mutations recently emerged as potential drivers of disease pathophysiology. Early synaptic problems in the excitatory cortical and striatal connections have been reported in HD, but the role of Htt protein in synaptic connectivity was unknown. Therefore, we investigated the role of Htt in synaptic connectivity in vivo by conditionally silencing Htt in the developing mouse cortex. When cortical Htt function was silenced, cortical and striatal excitatory synapses formed and matured at an accelerated pace through postnatal day 21 (P21). This exuberant synaptic connectivity was lost over time in the cortex, resulting in the deterioration of synapses by 5 weeks. Synaptic decline in the cortex was accompanied with layer- and region-specific reactive gliosis without cell loss. To determine whether the disease-causing poly-Q mutation in Htt affects synapse development, we next investigated the synaptic connectivity in a full-length knock-in mouse model of HD, the zQ175 mouse. Similar to the cortical conditional knock-outs, we found excessive excitatory synapse formation and maturation in the cortices of P21 zQ175, which was lost by 5 weeks. Together, our findings reveal that cortical Htt is required for the correct establishment of cortical and striatal excitatory circuits, and this function of Htt is lost when the mutant Htt is present.

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyltransferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons in vivo, and cell-free motility assays confirm a requirement of α-tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Together, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.