Faculty papers Farber Institute for Neurosciences

Glucose decouples intracellular Ca2+ activity from glucagon secretion in mouse pancreatic islet alpha-cells.

Sylvain J Le Marchand et al. — Wed, 19 Dec 2012 13:15:28 PST

The mechanisms of glucagon secretion and its suppression by glucose are presently unknown. This study investigates the relationship between intracellular calcium levels ([Ca(2+)](i)) and hormone secretion under low and high glucose conditions. We examined the effects of modulating ion channel activities on [Ca(2+)](i) and hormone secretion from ex vivo mouse pancreatic islets. Glucagon-secreting α-cells were unambiguously identified by cell specific expression of fluorescent proteins. We found that activation of L-type voltage-gated calcium channels is critical for α-cell calcium oscillations and glucagon secretion at low glucose levels. Calcium channel activation depends on K(ATP) channel activity but not on tetrodotoxin-sensitive Na(+) channels. The use of glucagon secretagogues reveals a positive correlation between α-cell [Ca(2+)](i) and secretion at low glucose levels. Glucose elevation suppresses glucagon secretion even after treatment with secretagogues. Importantly, this inhibition is not mediated by K(ATP) channel activity or reduction in α-cell [Ca(2+)](i). Our results demonstrate that glucose uncouples the positive relationship between [Ca(2+)](i) and secretory activity. We conclude that glucose suppression of glucagon secretion is not mediated by inactivation of calcium channels, but instead, it requires a calcium-independent inhibitory pathway.

Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington's disease knock-in mice.

Marina Kovalenko et al. — Wed, 19 Dec 2012 12:50:25 PST

The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.

Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer's disease-related tau phosphorylation via PAR-1.

Kanae Iijima-Ando et al. — Wed, 19 Dec 2012 12:42:18 PST

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.

Corticothalamic dysfunction and cognitive deficits in a mouse model of Alzheimer's disease

Brian Corbett et al. — Thu, 08 Nov 2012 07:31:53 PST

Presented at: Society for Neuroscience annual meeting in New Orleans, LA Oct 12-17, 2012

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with cognitive decline as well as a 5-10 fold increase in seizure incidence.

Although seizures were once thought to be secondary to disease, recent experiments suggest they may contribute to cognitive deficits early in disease progression. To investigate the underlying mechanisms, we use transgenic mice overexpressing human amyloid precursor protein (APP) bearing mutations that result in high levels of Aβ production. The type of seizures exhibited by APP mice suggests involvement of the corticothalamic network.

The corticothalamic network regulates a number of brain functions, including attention, learning and memory, cortical processing, and sleep maintenance which are all also affected in AD. To determine whether this network is indeed dysregulated in APP mice, we mapped activity in specific components of the corticothalamic network by assessing the expression and distribution of neuronal activity markers. We found decreased activity in an inhibitory thalamic control nucleus, and associated disinhibition of thalamic relay nuclei that project to hippocampal and cortical regions. APP mice also exhibited disturbances in sleep/wake patterns that were consistent with dysregulation of corticothalamic activity and were associated with hippocampal memory deficits. Thus, corticothalamic dysfunction may be a common denominator in AD pathophysiology and deserves further investigation.

Recommended citation: Corbett B, Zhang X, Zhao L, and Chin J (2012) Corticothalamic dysfunction and cognitive deficits in a mouse model of Alzheimer's disease. Soc for Neurosci Abstr 343.12.

Trk: a neuromodulator of age-specific behavioral and neurochemical responses to cocaine in mice.

Michelle Niculescu et al. — Fri, 13 Apr 2012 13:36:56 PDT

Responses to psychostimulants vary with age, but the molecular etiologies of these differences are largely unknown. The goal of the present research was to identify age-specific behavioral and molecular adaptations to cocaine and to elucidate the mechanisms involved therein. Postweanling, periadolescent, and adult male CD-1 mice were exposed to cocaine (20 mg/kg) for 7 d. The rewarding effects of cocaine were assessed, as were the response to a Trk antagonist and the regulation of dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32). Cocaine was rewarding in both periadolescent and adult mice using a conditioned place preference procedure. In contrast, postweanling mice failed to demonstrate significant cocaine-induced place preference. Because components of the neurotrophin system including brain-derived neurotrophic factor and TrkB are developmentally regulated, their role in the age-specific effects of cocaine was determined using the Trk receptor antagonist K252a. Postweanling mice that received K252a before daily cocaine showed a significant place preference to the cocaine-paired environment that was not seen in the absence of K252a. DARPP-32 protein levels were significantly upregulated in the lateral region of the caudate-putamen exclusively in postweanling mice after chronic cocaine. Daily pretreatment with K252a attenuated the induction of DARPP-32 in the postweanling striatum. These data indicate that Trk neurotransmission plays a role in age-specific behavioral and molecular responses to cocaine and concurrently modulates DARPP-32 levels.

Human amniotic fluid stem cells do not differentiate into dopamine neurons in vitro or after transplantation in vivo.

Angela E Donaldson et al. — Tue, 27 Mar 2012 10:57:52 PDT

Although embryonic stem (ES) cells can generate dopamine (DA) neurons that are potentially useful as a cell replacement therapy in Parkinson's disease (PD), associated ethical and practical concerns remain major stumbling blocks to their eventual use in humans. In this study, we examined human amniotic fluid stem (hAFS) cells derived from routine amniocenteses for their potential to give rise to DA neurons in vitro and following transplantation into the 6-hydroxydopamine-lesioned rat brain. We show that undifferentiated hAFS cells constitutively expressed mRNAs and proteins typical of stem cells but also cell derivatives of all three germ layers, including neural progenitors/neurons (nestin, beta-tubulin III, neurofilament). Additionally, these cells expressed mRNAs of an immature DA phenotype (Lmx1a, Pitx-3, Nurr1, Aldh1a1) but not the corresponding proteins. Importantly, treatment with DA differentiation factors using a variety of protocols did not further promote the development of fully differentiated DA neurons from hAFS cells. Thus, Lmx1a, Aldh1a1, AADC, TH, and DAT proteins were not detected in hAFS cells in culture or after transplantation into the PD rat brain. Moreover, by 3 weeks after implantation, there were no surviving AFS cells in the graft, likely as a result of an acute immunorejection response, as evidenced by the abundant presence of CD11+ macrophage/microglia and reactive GFAP+ astrocytes in the host brain. Taken together, these results suggest that further studies will be needed to improve differentiation procedures in culture and to prolong cell survival in vivo if hAFS cells are to be useful as replacement cells in PD.

Dopaminergic neurons derived from human induced pluripotent stem cells survive and integrate into 6-OHDA-lesioned rats.

Jingli Cai et al. — Fri, 23 Mar 2012 11:30:51 PDT

Cell replacement therapy could be an important treatment strategy for Parkinson's disease (PD), which is caused by the degeneration of dopamine neurons in the midbrain (mDA). The success of this approach greatly relies on the discovery of an abundant source of cells capable of mDAergic function in the brain. With the paucity of available human fetal tissue, efforts have increasingly focused on renewable stem cells. Human induced pluripotent stem (hiPS) cells offer great promise in this regard. If hiPS cells can be differentiated into authentic mDA neuron, hiPS could provide a potential autologous source of transplant tissue when generated from PD patients, a clear advantage over human embryonic stem (hES) cells. Here, we report that mDA neurons can be derived from a commercially available hiPS cell line, IMR90 clone 4, using a modified hES differentiation protocol established in our lab. These cells express all the markers (Lmx1a, Aldh1a1, TH, TrkB), follow the same mDA lineage pathway as H9 hES cells, and have similar expression levels of DA and DOPAC. Moreover, when hiPS mDA progenitor cells are transplanted into 6-OHDA-lesioned PD rats, they survive long term and many develop into bona fide mDA neurons. Despite their differentiation and integration into the brain, many Nestin+ tumor-like cells remain at the site of the graft. Our data suggest that as with hES cells, selecting the appropriate population of mDA lineage cells and eliminating actively dividing hiPS cells before transplantation will be critical for the future success of hiPS cell replacement therapy in PD patients.

Gene expression profile of neuronal progenitor cells derived from hESCs: activation of chromosome 11p15.5 and comparison to human dopaminergic neurons.

William J Freed et al. — Fri, 24 Feb 2012 10:40:26 PST

BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription.

METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined.

PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture.

CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.

Characterization of the Drosophila SLOWPOKE binding protein (SLOB) promoter

Amanda L. Sheldon et al. — Thu, 17 Nov 2011 11:43:46 PST

Drosophila SLOWPOKE (SLO) is a voltage and calcium dependent, large conductance potassium channel important for action potential repolarization, neuronal excitability, neurotransmitter release, and hormone secretion. SLO binding protein (SLOB) binds to and modulates SLO activity. We have shown previously that modulation of SLO by SLOB has profound effects on SLO channel currents, synaptic transmisison, and metabolism. Multiple isoforms of SLOB exist and are encoded by multiple transcripts; the isoforms are named based on their predicted protein molecular weights, in kilodaltons. In the Drosophila brain, SLOB57/51 proteins are expressed especially prominently in insulin producing neurons of the pars intercerebralis, while SLOB71/65 proteins are enriched in the lateral neurons that participate in the generation of circadian rhythms. Here we sought to determine the transcription initiation sites in the slob gene and investigated promoter elements responsible for expression of the different slob transcripts.

Amyloid-beta/Fyn–Induced Synaptic, Network, and Cognitive Impairments Depend on Tau Levels in Multiple Mouse Models of Alzheimer’s Disease

Erik D. Roberson et al. — Fri, 11 Feb 2011 09:01:18 PST

Alzheimer's disease (AD), the most common neurodegenerative disorder, is a growing public health problem and still lacks effective treatments. Recent evidence suggests that microtubule-associated protein tau may mediate amyloid-β peptide (Aβ) toxicity by modulating the tyrosine kinase Fyn.Weshowed previously that tau reduction prevents, and Fyn overexpression exacerbates, cognitive deficits in human amyloid precursor protein (hAPP) transgenic mice overexpressing Aβ. However, the mechanisms by which Aβ, tau, and Fyn cooperate in AD-related pathogenesis remain to be fully elucidated. Here we examined the synaptic and network effects of this pathogenic triad. Tau reduction prevented cognitive decline induced by synergistic effects of Aβ and Fyn. Tau reduction also prevented synaptic transmission and plasticity deficits in hAPP mice. Using electroencephalography to examine network effects, we found that tau reduction prevented spontaneous epileptiform activity in multiple lines of hAPP mice. Tau reduction also reduced the severity of spontaneous and chemically induced seizures in mice overexpressing both Aβ and Fyn. To better understand these protective effects, we recorded wholecell currents in acute hippocampal slices from hAPP mice with and without tau. hAPP mice with tau had increased spontaneous and evoked excitatory currents, reduced inhibitory currents, and NMDA receptor dysfunction. Tau reduction increased inhibitory currents and normalized excitation/inhibition balance and NMDA receptor-mediated currents in hAPP mice. Our results indicate that Aβ, tau, and Fyn jointly impair synaptic and network function and suggest that disrupting the copathogenic relationship between these factors could be of therapeutic benefit.

Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding.

Annat F Ikin et al. — Mon, 27 Sep 2010 08:31:41 PDT

ABSTRACT: BACKGROUND: Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as alpha-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. RESULTS: Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Rossner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPalpha. CONCLUSION: Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.

Similar promotion of Abeta1-42 fibrillogenesis by native apolipoprotein E epsilon3 and epsilon4 isoforms.

David Sweeney et al. — Mon, 27 Sep 2010 08:24:14 PDT

The apolipoprotein E epsilon4 allele contributes to the genetic susceptibility underlying a large proportion (~40-60%) of typical, sporadic Alzheimer disease. Apolipoprotein E deficient mice made transgenic for human apolipoprotein E epsilon4 accumulate excess cerebral amyloid when compared to similarly prepared mice expressing human apolipoprotein E epsilon3. Therefore, it is important to search for relevant interactions(s) between apolipoprotein E epsilon4 and Abeta in order to clarify the biological role for apolipoprotein E epsilon4 in Alzheimer disease. Using a thioflavine T (ThT)-based assay, we have investigated the effects of native human apolipoprotein E isoforms on the kinetics of Abeta fibrillogenesis. No obvious profibrillogenic activity was detected in Abeta1-40-based assays of any native apolipoprotein E isoform. However, when ThT assays were repeated using Abeta1-42, modest, but statistically significant, profibrillogenic activity was detected in both apolipoprotein E epsilon3- and apolipoprotein E epsilon4-containing media and was similar in magnitude for the two isoforms. These data demonstrate that native apolipoprotein E possesses "pathological chaperone"-type activity for Abeta: in other words, the data indicate that a chaperone-like misfolding reaction can occur between native apolipoprotein E and Abeta. However, the equipotent activities of the apolipoprotein E epsilon3 and epsilon4 isoforms suggests the possibility that either extended co-incubation of apolipoprotein E and Abeta, or, perhaps, the inclusion in the reaction of other fibrillogenesis-modulation co-factors (such as metal ions, or inflammatory mediators such as reactive oxygen species, alpha2-macroglobulin, apolipoprotein J, etc.) may be required for modeling in vitro the apolipoprotein E-isoform-specific-regulation of extracellular Abeta accumulation that occurs in vivo. Alternatively, other events, such as differential apolipoprotein E-isoform-mediated clearance of Abeta or of apolipoprotein E/Abeta complexes may underlie apolipoprotein E-isoform-dependent Abeta accumulation.