MANF regulates hypothalamic control of food intake and body weight.

The hypothalamus has a vital role in controlling food intake and energy homeostasis; its activity is modulated by neuropeptides and endocrine factors. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neurotrophic factor that is also localized in the endoplasmic reticulum (ER) in neurons. Here we show that MANF is highly enriched in distinct nuclei of the mouse hypothalamus, and that MANF expression in the hypothalamus is upregulated in response to fasting. Increasing or decreasing hypothalamic MANF protein levels causes hyperphagia or hypophagia, respectively. Moreover, MANF triggers hypothalamic insulin resistance by enhancing the ER localization and activity of PIP4k2b, a kinase known to regulate insulin signaling. Our findings indicate that MANF influences food intake and body weight by modulating hypothalamic insulin signaling.MANF is a neurotrophic factor that is secreted but also mediates the unfolded protein response acting intracellularly. Here, the authors show that MANF expression in the brain is influenced by nutritional cues, and hypothalamic MANF influences food intake and systemic energy homeostasis.

DYRK1A regulates Hap1-Dcaf7/WDR68 binding with implication for delayed growth in Down syndrome.

Huntingtin-associated protein 1 (Hap1) is known to be critical for postnatal hypothalamic function and growth. Hap1 forms stigmoid bodies (SBs), unique neuronal cytoplasmic inclusions of unknown function that are enriched in hypothalamic neurons. Here we developed a simple strategy to isolate the SB-enriched fraction from mouse brain. By analyzing Hap1 immunoprecipitants from this fraction, we identified a Hap1-interacting SB component, DDB1 and CUL4 associated factor 7 (Dcaf7)/WD40 repeat 68 (WDR68), whose protein level and nuclear translocation are regulated by Hap1. Moreover, we found that Hap1 bound Dcaf7 competitively in cytoplasm with dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), a protein implicated in Down syndrome (DS). Depleting Hap1 promoted the DYRK1A-Dcaf7 interaction and increased the DYRK1A protein level. Transgenic DS mice overexpressing DYRK1A showed reduced Hap1-Dcaf7 association in the hypothalamus. Furthermore, the overexpression of DYRK1A in the hypothalamus led to delayed growth in postnatal mice, suggesting that DYRK1A regulates the Hap1-Dcaf7 interaction and postnatal growth and that targeting Hap1 or Dcaf7 could ameliorate growth retardation in DS.

N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy.

The Huntington's disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1-208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD.

Ablation of huntingtin in adult neurons is nondeleterious but its depletion in young mice causes acute pancreatitis.

The Huntington's disease (HD) protein, huntingtin (HTT), is essential for early development. Because suppressing the expression of mutant HTT is an important approach to treat the disease, we must first understand the normal function of Htt in adults versus younger animals. Using inducible Htt knockout mice, we found that Htt depletion does not lead to adult neurodegeneration or animal death at >4 mo of age, which was also verified by selectively depleting Htt in neurons. On the other hand, young Htt KO mice die at 2 mo of age of acute pancreatitis due to the degeneration of pancreatic acinar cells. Importantly, Htt interacts with the trypsin inhibitor, serine protease inhibitor Kazal-type 3 (Spink3), to inhibit activation of digestive enzymes in acinar cells in young mice, and transgenic HTT can rescue the early death of Htt KO mice. These findings point out age- and cell type-dependent vital functions of Htt and the safety of knocking down neuronal Htt expression in adult brains as a treatment.

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice.

In polyglutamine (polyQ) diseases, large polyQ repeats cause juvenile cases with different symptoms than those of adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knockin mouse models of spinal cerebellar ataxia-17 (SCA17), we found that a large polyQ (105 glutamines) in the TATA-box-binding protein (TBP) preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP's interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases.

Therapeutic Effect of Berberine on Huntington's Disease Transgenic Mouse Model.

Huntington disease (HD) represents a family of neurodegenerative diseases that are caused by misfolded proteins. The misfolded proteins accumulate in the affected brain regions in an age-dependent manner to cause late-onset neurodegeneration. Transgenic mouse models expressing the HD protein, huntingtin, have been widely used to identify therapeutics that may retard disease progression. Here we report that Berberine (BBR), an organic small molecule isolated from plants, has protective effects on transgenic HD (N171-82Q) mice. We found that BBR can reduce the accumulation of mutant huntingtin in cultured cells. More importantly, when given orally, BBR could effectively alleviate motor dysfunction and prolong the survival of transgenic N171-82Q HD mice. We found that BBR could promote the degradation of mutant huntingtin by enhancing autophagic function. Since BBR is an orally-taken drug that has been safely used to treat a number of diseases, our findings suggest that BBR can be tested on different HD animal models and HD patients to further evaluate its therapeutic effects.

Mutant huntingtin downregulates myelin regulatory factor-mediated myelin gene expression and affects mature oligodendrocytes.

Growing evidence indicates that non-neuronal mutant huntingtin toxicity plays an important role in Huntington's disease (HD); however, whether and how mutant huntingtin affects oligodendrocytes, which are vitally important for neural function and axonal integrity, remains unclear. We first verified the presence of mutant huntingtin in oligodendrocytes in HD140Q knockin mice. We then established transgenic mice (PLP-150Q) that selectively express mutant huntingtin in oligodendrocytes. PLP-150Q mice show progressive neurological symptoms and early death, as well as age-dependent demyelination and reduced expression of myelin genes that are downstream of myelin regulatory factor (MYRF or MRF), a transcriptional regulator that specifically activates and maintains the expression of myelin genes in mature oligodendrocytes. Consistently, mutant huntingtin binds abnormally to MYRF and affects its transcription activity. Our findings suggest that dysfunction of mature oligodendrocytes is involved in HD pathogenesis and may also make a good therapeutic target.

TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

Age-dependent decrease in chaperone activity impairs MANF expression, leading to Purkinje cell degeneration in inducible SCA17 mice.

Although protein-misfolding-mediated neurodegenerative diseases have been linked to aging, how aging contributes to selective neurodegeneration remains unclear. We established spinocerebellar ataxia 17 (SCA17) knockin mice that inducibly express one copy of mutant TATA box binding protein (TBP) at different ages by tamoxifen-mediated Cre recombination. We find that more mutant TBP accumulates in older mouse and that this accumulation correlates with age-related decreases in Hsc70 and chaperone activity. Consistently, older SCA17 mice experienced earlier neurological symptom onset and more severe Purkinje cell degeneration. Mutant TBP shows decreased association with XBP1s, resulting in the reduced transcription of mesencephalic astrocyte-derived neurotrophic factor (MANF), which is enriched in Purkinje cells. Expression of Hsc70 improves the TBP-XBP1s interaction and MANF transcription, and overexpression of MANF ameliorates mutant TBP-mediated Purkinje cell degeneration via protein kinase C (PKC)-dependent signaling. These findings suggest that the age-related decline in chaperone activity affects polyglutamine protein function that is important for the viability of specific types of neurons.

Synaptic mutant huntingtin inhibits synapsin-1 phosphorylation and causes neurological symptoms.

Many genetic mouse models of Huntington's disease (HD) have established that mutant huntingtin (htt) accumulates in various subcellular regions to affect a variety of cellular functions, but whether and how synaptic mutant htt directly mediates HD neuropathology remains to be determined. We generated transgenic mice that selectively express mutant htt in the presynaptic terminals. Although it was not overexpressed, synaptic mutant htt caused age-dependent neurological symptoms and early death in mice as well as defects in synaptic neurotransmitter release. Mass spectrometry analysis of synaptic fractions and immunoprecipitation of synapsin-1 from HD CAG150 knockin mouse brains revealed that mutant htt binds to synapsin-1, a protein whose phosphorylation is critical for neurotransmitter release. We found that polyglutamine-expanded exon1 htt binds to the C-terminal region of synapsin-1 to reduce synapsin-1 phosphorylation. Our findings point to a critical role for synaptic htt in the neurological symptoms of HD, providing a new therapeutic target.

Loss of Ahi1 affects early development by impairing BM88/Cend1-mediated neuronal differentiation.

Mutations in the Abelson helper integration site-1 (AHI1) gene result in N-terminal Ahi1 fragments and cause Joubert syndrome, an autosomal recessive brain malformation disorder associated with delayed development. How AHI1 mutations lead to delayed development remains unclear. Here we report that full-length, but not N-terminal, Ahi1 binds Hap1, a huntingtin-associated protein that is essential for the postnatal survival of mice and that this binding is regulated during neuronal differentiation by nerve growth factor. Nerve growth factor induces dephosphorylation of Hap1A and decreases its association with Ahi1, correlating with increased Hap1A distribution in neurite tips. Consistently, Ahi1 associates with phosphorylated Hap1A in cytosolic, but not in synaptosomal, fractions isolated from mouse brain, suggesting that Ahi1 functions mainly in the soma of neurons. Mass spectrometry analysis of cytosolic Ahi1 immunoprecipitates reveals that Ahi1 also binds Cend1 (cell cycle exit and neuronal differentiation protein 1)/BM88, a neuronal protein that mediates neuronal differentiation and is highly expressed in postnatal mouse brain. Loss of Ahi1 reduces the levels of Cend1 in the hypothalamus of Ahi1 KO mice, which show retarded growth during postnatal days. Overexpressed Ahi1 can stabilize Cend1 in cultured cells. Furthermore, overexpression of Cend1 can rescue the neurite extension defects of hypothalamic neurons from Ahi1 KO mice. Our findings suggest that Cend1 is involved in Ahi1-associated hypothalamic neuronal differentiation in early development, giving us fresh insight into the mechanism behind the delayed development in Joubert syndrome.