Steven Finkbeiner

Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.

Huntingtons disease is caused by an abnormal polyglutamine expansion within the protein huntingtin and is characterized by microscopic inclusion bodies of aggregated huntingtin and by the death of selected types of neuron. Whether inclusion bodies are pathogenic, incidental or a beneficial coping response is controversial. To resolve this issue we have developed an automated microscope that returns to precisely the same neuron after arbitrary intervals, even after cells have been removed from the microscope stage. Here we show, by survival analysis, that neurons die in a time-independent fashion but one that is dependent on mutant huntingtin dose and polyglutamine expansion; many neurons die without forming an inclusion body. Rather, the amount of diffuse intracellular huntingtin predicts whether and when inclusion body formation or death will occur. Surprisingly, inclusion body formation predicts improved survival and leads to decreased levels of mutant huntingtin elsewhere in a neuron. Thus, inclusion body formation can function as a coping response to toxic mutant huntingtin.

Huntingtin Acts in the Nucleus to Induce Apoptosis but Death Does Not Correlate with the Formation of Intranuclear Inclusions

The mechanisms by which mutant huntingtin induces neurodegeneration were investigated using a cellular model that recapitulates features of neurodegeneration seen in Huntingtons disease. When transfected into cultured striatal neurons, mutant huntingtin induces neurodegeneration by an apoptotic mechanism. Antiapoptotic compounds or neurotrophic factors protected neurons against mutant huntingtin. Blocking nuclear localization of mutant huntingtin suppressed its ability to form intranuclear inclusions and to induce neurodegeneration. However, the presence of inclusions did not correlate with huntingtin-induced death. The exposure of mutant huntingtin-transfected striatal neurons to conditions that suppress the formation of inclusions resulted in an increase in mutant huntingtin-induced death. These findings suggest that mutant huntingtin acts within the nucleus to induce neurodegeneration. However, intranuclear inclusions may reflect a cellular mechanism to protect against huntingtin-induced cell death.

Ca2+ Influx Regulates BDNF Transcription by a CREB Family Transcription Factor-Dependent Mechanism

CREB is a transcription factor implicated in the control of adaptive neuronal responses. Although one function of CREB in neurons is believed to be the regulation of genes whose products control synaptic function, the targets of CREB that mediate synaptic function have not yet been identified. This report describes experiments demonstrating that CREB or a closely related protein mediates Ca2+-dependent regulation of BDNF, a neurotrophin that modulates synaptic activity. In cortical neurons, Ca2+ influx triggers phosphorylation of CREB, which by binding to a critical Ca2+ response element (CRE) within the BDNF gene activates BDNF transcription. Mutation of the BDNF CRE or an adjacent novel regulatory element as well as a blockade of CREB function resulted in a dramatic loss of BDNF transcription. These findings suggest that a CREB family member acts cooperatively with an additional transcription factor(s) to regulate BDNF transcription. We conclude that the BDNF gene is a CREB family target whose protein product functions at synapses to control adaptive neuronal responses.

Aberrant Excitatory Neuronal Activity and Compensatory Remodeling of Inhibitory Hippocampal Circuits in Mouse Models of Alzheimer's Disease

Neural network dysfunction may play an important role in Alzheimers disease (AD). Neuronal circuits vulnerable to AD are also affected in human amyloid precursor protein (hAPP) transgenic mice. hAPP mice with high levels of amyloid-beta peptides in the brain develop AD-like abnormalities, including cognitive deficits and depletions of calcium-related proteins in the dentate gyrus, a region critically involved in learning and memory. Here, we report that hAPP mice have spontaneous nonconvulsive seizure activity in cortical and hippocampal networks, which is associated with GABAergic sprouting, enhanced synaptic inhibition, and synaptic plasticity deficits in the dentate gyrus. Many Abeta-induced neuronal alterations could be simulated in nontransgenic mice by excitotoxin challenge and prevented in hAPP mice by blocking overexcitation. Aberrant increases in network excitability and compensatory inhibitory mechanisms in the hippocampus may contribute to Abeta-induced neurological deficits in hAPP mice and, possibly, also in humans with AD.

The IGF-1/Akt Pathway Is Neuroprotective in Huntington's Disease and Involves Huntingtin Phosphorylation by Akt

In the search for neuroprotective factors in Huntingtons disease, we found that insulin growth factor 1 via activation of the serine/threonine kinase Akt/PKB is able to inhibit neuronal death specifically induced by mutant huntingtin containing an expanded polyglutamine stretch. The IGF-1/Akt pathway has a dual effect on huntingtin-induced toxicity, since activation of this pathway also results in a decrease in the formation of intranuclear inclusions of mutant huntingtin. We demonstrate that huntingtin is a substrate of Akt and that phosphorylation of huntingtin by Akt is crucial to mediate the neuroprotective effects of IGF-1. Finally, we show that Akt is altered in Huntingtons disease patients. Taken together, these results support a potential role of the Akt pathway in Huntingtons disease.

CREB couples neurotrophin signals to survival messages.

There are several interacting pathways for survival, death, bcl-2 transcription, and CREB Ser-133 phosphorylation. Some of this richness is described below. For example, although CREB-dependent activation of bcl-2 transcription is an attractive mechanism to explain neurotrophin-induced survival, the contribution of this pathway to programmed cell death in vivo remains to be established. In vivo, the absence of NGF, its cognate receptor TrkA, or bcl-2 causes overlapping populations of neurons to die. However, significant neurodegeneration in bcl-2−/− mice appears to occur after a time point (P10) when the majority of these neurons are lost in NGF−/− or TrkA−/− mice, and loss of cerebellar granule neurons in bcl-2−/− mice has not been reported (Michaelidis et al. 1996xMichaelidis, T.M, Sendtner, M, Cooper, J.D, Airaksinen, M.S, Holtmann, B, Meyer, M, and Thoenen, H. Neuron. 1996; 17: 75–89Abstract | Full Text | Full Text PDF | PubMed | Scopus (218)See all ReferencesMichaelidis et al. 1996). Moreover, BDNF is capable of promoting the survival of motoneurons following axotomy, even in bcl-2−/− mice (Michaelidis et al. 1996xMichaelidis, T.M, Sendtner, M, Cooper, J.D, Airaksinen, M.S, Holtmann, B, Meyer, M, and Thoenen, H. Neuron. 1996; 17: 75–89Abstract | Full Text | Full Text PDF | PubMed | Scopus (218)See all ReferencesMichaelidis et al. 1996). Thus, additional neurotrophin-dependent signal transduction pathways and CREB target genes may mediate neuronal survival. To test this possibility, it would be interesting to determine if CREB-VP16 can promote the survival of sympathetic neurons or cerebellar granule cells from bcl-2−/− mice.Support for the existence of additional transcription-dependent survival pathways has appeared recently (Figure 2Figure 2). Akt/PKB mediates neuronal survival partly through phosphorylation and inhibition of FKHRL1, a member of the Forkhead family of transcription factors (reviewed by Datta et al. 1999xDatta, S.R, Brunet, A, and Greenberg, M.E. Genes Dev. 1999; 13: 2905–2927Crossref | PubMed | Scopus (3200)See all ReferencesDatta et al. 1999). FKHRL1 phosphorylation prevents it from translocating to the nucleus and upregulating the proapoptotic protein Fas ligand. Recently, the ability of Ca2+ influx to promote granule cell survival was shown to depend partly on the activation of MEF-2-dependent transcription (Mao et al. 1999xMao, Z, Bonni, A, Xia, F, Nadal-Vicens, M, and Greenberg, M.E. Science. 1999; 286: 785–790Crossref | PubMed | Scopus (363)See all ReferencesMao et al. 1999). Taken together, these results establish a commonly held idea—that a major mechanism by which extracellular stimuli such as neurotrophins and neuronal activity regulate the survival of specific populations of developing neurons is through the activation or repression of gene transcription. Thus, a major focus for future research will be to identify the critical target genes that mediate survival and to discover how factors, such as CREB, regulate their transcription.For some survival genes, CREB may need to interact with other promoter-bound factors to achieve physiologic patterns of gene expression. The bcl-2 gene contains an upstream response element (URE) that is adjacent but upstream of the CRE (Wilson et al. 1996xWilson, B.E, Mochon, E, and Boxer, L.M. Mol. Cell Biol. 1996; 16: 5546–5556Crossref | PubMedSee all ReferencesWilson et al. 1996). Although an isolated URE cannot mediate bcl-2 expression, the URE cooperates with the adjacent CRE to attain levels of bcl-2 reporter gene expression that are double those achieved with the CRE alone (Wilson et al. 1996xWilson, B.E, Mochon, E, and Boxer, L.M. Mol. Cell Biol. 1996; 16: 5546–5556Crossref | PubMedSee all ReferencesWilson et al. 1996). In this regard, bcl-2 is organized similar to BDNF, another CREB-regulated gene that mediates neuronal survival. Ca2+-stimulated BDNF transcription depends on interactions within a BDNF promoter between CRE-bound factors and proteins that are bound to an adjacent URE (reviewed by Shieh and Ghosh 1999xShieh, B and Ghosh, A. J. Neurobiol. 1999; 41: 127–134Crossref | PubMed | Scopus (118)See all ReferencesShieh and Ghosh 1999). However, the identity of upstream factors that cooperate with CREB to regulate BDNF or bcl-2 transcription and the significance of the URE/CRE organization remain unknown.Response elements in addition to the URE and CRE may also mediate NGF-induced bcl-2 transcription (Wilson et al. 1996xWilson, B.E, Mochon, E, and Boxer, L.M. Mol. Cell Biol. 1996; 16: 5546–5556Crossref | PubMedSee all ReferencesWilson et al. 1996). Downstream of the known CRE, bcl-2 contains a negative regulatory element that binds the tumor suppressor p53. Kaplan and colleagues recently showed that NGF activation of the Ras/ERK pathway regulates the survival of sympathetic neurons partly by suppressing the p53-mediated cell death pathway (Mazzoni et al. 1999xMazzoni, I.E, Sad, F.A, Aloyz, R, Miller, F.D, and Kaplan, D. J. Neurosci. 1999; 19: 9716–9727PubMedSee all ReferencesMazzoni et al. 1999). Thus, NGF and other neurotrophins might control the expression of bcl-2 positively through CREB and negatively through p53 suppression.The discovery that CREB is a critical regulator of NGF-induced bcl-2 transcription and neuronal survival raises the interesting possibility that CREB and related family members might play a general role in mediating stimulus-dependent cell survival. The critical Ser-133 on CREB that is phosphorylated by RSK-2 is also a target of many other signal transduction pathways besides the Ras/ERK pathway. Several of these pathways, such as protein kinase A (PKA), PKC, and CaMKIV, are activated by extracellular stimuli that promote the survival of some neuronal and nonneuronal cell populations. The observation that mice lacking CREB mostly survive until birth and apparently exhibit limited anatomical and physiological defects could be construed as evidence against a broad role for CREB in survival (Rudolph et al. 1998xRudolph, D, Tafuri, A, Gass, P, Hammerling, G.J, Arnold, B, and Shutz, G. Proc. Natl. Acad. Sci. USA. 1998; 95: 4481–4486Crossref | PubMed | Scopus (233)See all ReferencesRudolph et al. 1998). However, other CREB family members probably substitute for the absence of CREB in these mice (Rudolph et al. 1998xRudolph, D, Tafuri, A, Gass, P, Hammerling, G.J, Arnold, B, and Shutz, G. Proc. Natl. Acad. Sci. USA. 1998; 95: 4481–4486Crossref | PubMed | Scopus (233)See all ReferencesRudolph et al. 1998).CREB has already been implicated in mechanisms of activity-dependent synaptic plasticity (reviewed by Silva et al. 1998xSilva, A.J, Kogan, J.H, Frankland, P.W, and Kida, S. Annu. Rev. Neurosci. 1998; 21: 127–148Crossref | PubMed | Scopus (897)See all ReferencesSilva et al. 1998). Now, the reports from the Ginty and Greenberg laboratories show that CREB also regulates neuronal survival. How does the same transcription factor mediate apparently distinct biological functions? CREB is a single component of a complex of promoter-bound enhancing factors. Whether a particular gene is transcribed in response to a signal is determined by the factors bound to the genes promoter and the activity of the pathways that regulate the complex. Therefore, specificity may reflect the activity of parallel pathways which regulate factors that cooperate with CREB. However, it is tempting to speculate that the signal transduction pathways and programs of gene expression involved in activity-dependent survival and activity-dependent plasticity may largely overlap. For example, neurotrophic factors, extracellular Ca2+ influx, Ras/ERK pathway activation, and now CREB-dependent transcription have been assigned dual functions, regulating both neuronal survival and synaptic plasticity (reviewed by Curtis and Finkbeiner 1999xCurtis, J and Finkbeiner, S. J. Neurosci. Res. 1999; 58: 88–95Crossref | PubMed | Scopus (99)See all ReferencesCurtis and Finkbeiner 1999). A molecular connection between pathways that regulate survival and plasticity could lend support to the idea that some neurodegenerative disorders may ultimately prove to be a failure of plasticity (Mesulam 1999xMesulam, M.-M. Neuron. 1999; 24: 521–529Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesMesulam 1999).*E-mail: sfinkbeiner@gladstone.ucsf.edu.

Direct Membrane Association Drives Mitochondrial Fission by the Parkinson Disease-associated Protein α-Synuclein

The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease.

Tau Reduction Prevents Aβ-Induced Defects in Axonal Transport

A mechanism for the protective effects of tau reduction in mouse models of Alzheimer’s disease. Amyloid-β (Aβ) peptides, derived from the amyloid precursor protein, and the microtubule-associated protein tau are key pathogenic factors in Alzheimer’s disease (AD). How exactly they impair cognitive functions is unknown. We assessed the effects of Aβ and tau on axonal transport of mitochondria and the neurotrophin receptor TrkA, cargoes that are critical for neuronal function and survival and whose distributions are altered in AD. Aβ oligomers rapidly inhibited axonal transport of these cargoes in wild-type neurons. Lowering tau levels prevented these defects without affecting baseline axonal transport. Thus, Aβ requires tau to impair axonal transport, and tau reduction protects against Aβ-induced axonal transport defects.

Calcium waves in astrocytes-filling in the gaps.

Stimulus-evoked cellular responses are sometimes organized in the form of propagating waves of cytoplasmic Ca2+ increase. Ca2+ waves can be elicited in cultured astrocytes by the neurotransmitter glutamate; however, the propagation mechanism is unknown. Here, qualitative and quantitative features of propagation suggest that astrocytic Ca2+ waves are mediated by an intracellular signal that crosses intercellular junctions. The role of gap junctions in cell-cell Ca2+ wave propagation was specifically tested. Functional gap junctions were demonstrated using a noninvasive fluorescence recovery method and the gap junction blockers halothane and octanol. Gap junction closure prevented intracellular waves from propagating between cells without affecting the velocity of the intracellular wave itself. The pivotal role played by the gap junction creates the potential for dynamic changes in glial connectivity and long-range glial signaling.

Induced Pluripotent Stem Cells from Patients with Huntington’s Disease : Show CAG Repeat-Expansion-Associated Phenotypes

Huntingtons disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, The HD Consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG-repeat-expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease-associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a human stem cell platform for screening new candidate therapeutics.