TGF-ß/Smad3 Signalling Modulates GABA Neurotransmission: Implications in Parkinson's Disease.

γ-Aminobutiryc acid (GABA) is found extensively in different brain nuclei, including parts involved in Parkinson's disease (PD), such as the basal ganglia and hippocampus. In PD and in different models of the disorder, an increase in GABA neurotransmission is observed and may promote bradykinesia or L-Dopa-induced side-effects. In addition, proteins involved in GABAA receptor (GABAAR) trafficking, such as GABARAP, Trak1 or PAELR, may participate in the aetiology of the disease. TGF-ß/Smad3 signalling has been associated with several pathological features of PD, such as dopaminergic neurodegeneration; reduction of dopaminergic axons and dendrites; and α-synuclein aggregation. Moreover, TGF-ß/Smad3 intracellular signalling was recently shown to modulate GABA neurotransmission in the context of parkinsonism and cognitive alterations. This review provides a summary of GABA neurotransmission and TGF-ß signalling; their implications in PD; and the regulation of GABA neurotransmission by TGF-ß/Smad3. There appear to be new possibilities to develop therapeutic approaches for the treatment of PD using GABA modulators.

Smad3 deficiency inhibits dentate gyrus LTP by enhancing GABAA neurotransmission.

Transforming growth factor-ß signaling through intracellular Smad3 has been implicated in Parkinson's disease (PD) and it fulfills an important role in the neurogenesis and synaptic plasticity that occurs in the adult dentate gyrus (DG). The long-term potentiation (LTP) induced in the DG by high-frequency stimulation of the medial perforant pathway is abolished in the DG of Smad3-deficient mice, but not in the CA1 hippocampal region. Here, we show that NMDA- and AMPA-type glutamate receptors do not participate in the inhibition of LTP associated with Smad3 deficiency. Moreover, there is no difference in the hippocampal GAD65 and GAD67 content, suggesting that GABA biosynthesis remains unaffected. Increased conductance and higher action potential firing thresholds were evident in intracellular recordings of granule cells from Smad3 deficient mice. Interestingly, phasic and tonic GABAA receptor (GABAA R)-mediated neurotransmission is enhanced in the DG of Smad3-deficient mice, and LTP induction can be rescued by inhibiting GABAA R with picrotoxin. Hence, Smad3 signaling in the DG appears to be necessary to induce LTP by regulating GABAA neurotransmission, suggesting a central role of this intracellular signaling pathway in the hippocampal brain plasticity related to learning and memory. Smad3 deficient mice represent a new and interesting model of Parkinson's disease, displaying hippocampal dysfunctions that include decreased neurogenesis and the failure to induce LTP in the dentate gyrus. Here we show that Smad3 deficiency inhibits LTP induction by enhancing phasic and tonic GABAA receptor-mediated neurotransmission, while LTP induction can be rescued with a GABAA receptor antagonist. Alteration of GABA neurotransmission is thought to produce hippocampal cognitive dysfunction in Down's syndrome or Alzheimer's disease, and here we provide new insights into the hippocampal changes in an animal model of Parkinson's disease.

Models of α-synuclein aggregation in Parkinson's disease.

Parkinson's disease (PD) is not only characterized by motor disturbances but also, by cognitive, sensory, psychiatric and autonomic dysfunction. It has been proposed that some of these symptoms might be related to the widespread pathology of α-synuclein (α-syn) aggregation in different nuclei of the central and peripheral nervous system. However, the pathogenic formation of α-syn aggregates in different brain areas of PD patients is poorly understood. Most experimental models of PD are valuable to assess specific aspects of its pathogenesis, such as toxin-induced dopaminergic neurodegeneration. However, new models are required that reflect the widespread and progressive formation of α-syn aggregates in different brain areas. Such α-syn aggregation is induced in only a few animal models, for example perikaryon inclusions are found in rats administered rotenone, aggregates with a neuritic morphology develop in mice overexpressing either mutated or wild-type α-syn, and in Smad3 deficient mice, aggregates form extensively in the perikaryon and neurites of specific brain nuclei. In this review we focus on α-syn aggregation in the human disorder, its genetics and the availability of experimental models. Indeed, evidences show that dopamine (DA) metabolism may be related to α-syn and its conformational plasticity, suggesting an interesting link between the two pathological hallmarks of PD: dopaminergic neurodegeneration and Lewy body (LB) formation.

Smad3 is required for the survival of proliferative intermediate progenitor cells in the dentate gyrus of adult mice.

BACKGROUND: New neurons are continuously being generated in the adult hippocampus, a phenomenon that is regulated by external stimuli, such as learning, memory, exercise, environment or stress. However, the molecular mechanisms underlying neuron production and how they are integrated into existing circuits under such physiological conditions remain unclear. Indeed, the intracellular modulators that transduce the extracellular signals are not yet fully understood. RESULTS: We show that Smad3, an intracellular molecule involved in the transforming growth factor (TGF)-ß signaling cascade, is strongly expressed by granule cells in the dentate gyrus (DG) of adult mice, although the loss of Smad3 in null mutant mice does not affect their survival. Smad3 is also expressed by adult progenitor cells in the subgranular zone (SGZ) and more specifically, it is first expressed by Type 2 cells (intermediate progenitor cells). Its expression persists through the distinct cell stages towards that of the mature neuron. Interestingly, proliferative intermediate progenitor cells die in Smad3 deficiency, which is associated with a large decrease in the production of newborn neurons in Smad3 deficient mice. Smad3 signaling appears to influence adult neurogenesis fulfilling distinct roles in the rostral and mid-caudal regions of the DG. In rostral areas, Smad3 deficiency increases proliferation and promotes the cell cycle exit of undifferentiated progenitor cells. By contrast, Smad3 deficiency impairs the survival of newborn neurons in the mid-caudal region of the DG at early proliferative stages, activating apoptosis of intermediate progenitor cells. Furthermore, long-term potentiation (LTP) after high frequency stimulation (HFS) to the medial perforant path (MPP) was abolished in the DG of Smad3-deficient mice. CONCLUSIONS: These data show that endogenous Smad3 signaling is central to neurogenesis and LTP induction in the adult DG, these being two forms of hippocampal brain plasticity related to learning and memory that decline with aging and as a result of neurological disorders.

Dopamine and α-synuclein dysfunction in Smad3 null mice.

BACKGROUND: Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra (SN). Transforming growth factor-ß1 (TGF-ß1) levels increase in patients with PD, although the effects of this increment remain unclear. We have examined the mesostriatal system in adult mice deficient in Smad3, a molecule involved in the intracellular TGF-ß1 signalling cascade. RESULTS: Striatal monoamine oxidase (MAO)-mediated dopamine (DA) catabolism to 3,4-dihydroxyphenylacetic acid (DOPAC) is strongly increased, promoting oxidative stress that is reflected by an increase in glutathione levels. Fewer astrocytes are detected in the ventral midbrain (VM) and striatal matrix, suggesting decreased trophic support to dopaminergic neurons. The SN of these mice has dopaminergic neuronal degeneration in its rostral portion, and the pro-survival Erk1/2 signalling is diminished in nigra dopaminergic neurons, not associated with alterations to p-JNK or p-p38. Furthermore, inclusions of α-synuclein are evident in selected brain areas, both in the perikaryon (SN and paralemniscal nucleus) or neurites (motor and cingulate cortices, striatum and spinal cord). Interestingly, these α-synuclein deposits are detected with ubiquitin and P(S129)-α-synuclein in a core/halo cellular distribution, which resemble those observed in human Lewy bodies (LB). CONCLUSIONS: Smad3 deficiency promotes strong catabolism of DA in the striatum (ST), decrease trophic and astrocytic support to dopaminergic neurons and may induce α-synuclein aggregation, which may be related to early parkinsonism. These data underline a role for Smad3 in α-synuclein and DA homeostasis, and suggest that modulatory molecules of this signalling pathway should be evaluated as possible neuroprotective agents.

TIMP-1 abolishes MMP-9-dependent long-lasting long-term potentiation in the prefrontal cortex.

BACKGROUND: Understanding of the molecular mechanisms of prefrontal cortex (PFC) plasticity is important for developing new treatment strategies for mental disorders such as depression and schizophrenia. Long-term potentiation (LTP) is a valid model for synaptic plasticity. The extracellular proteolytic system composed of matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) has recently been shown to play major role in the hippocampal plasticity. METHODS: We tested whether induction of hippocampal-prefrontal LTP results in accumulation of tissue inhibitor of MMP-1, TIMP-1 mRNA, in the PFC of rats and whether adenovirally driven overexpression of TIMP-1 affects LTP. Additional study of slices was done with a specific MMP-9 inhibitor. RESULTS: The TIMP-1 is induced in the rat medial PFC by stimuli evoking late LTP; its overexpression blocks the gelatinolytic activity of the MMP family; its overexpression before tetanization blocks late LTP in vivo; and MMP-9 inhibitor prevents late LTP in vitro. CONCLUSIONS: We suggest a novel extracellular mechanism of late LTP in the PFC, engaging TIMP-1-controlled proteolysis as an element of information integration. Our results may also be meaningful to an understanding of mental diseases and development of new treatment strategies that are based on extracellular mechanisms of synaptic plasticity.

Ca2+/calmodulin kinase kinase alpha is dispensable for brain development but is required for distinct memories in male, though not in female, mice.

In neurons, the Ca(2+)/calmodulin (CaM) kinase cascade transduces Ca(2+) signaling into gene transcription. The CaM kinase cascade is known to be important for brain development as well as memory formation in adult brain, although the functions of some cascade members remain unknown. Here we have generated null and hypomorphic mutants to study the physiological role of CaM kinase kinase alpha (CaMKKalpha), which phosphorylates and activates both CaM kinase I (CaMKI) and CaMKIV, the output kinases of the cascade. We show that CaMKKalpha is dispensable for brain development and long-term potentiation in adult hippocampal CA1 synapses. We find that CaMKKalpha is required for hippocampus-dependent contextual fear memory, but not spatial memory, formation. Surprisingly, CaMKKalpha is important for contextual fear memory formation in males but not in females. We show that in male mice, contextual fear conditioning induces up-regulation of hippocampal mRNA expression of brain-derived neurotrophic factor (BDNF) in a way that requires CaMKKalpha, while in female mice, contextual fear conditioning induces down-regulation of hippocampal BDNF mRNA expression that does not require CaMKKalpha. Additionally, we demonstrate sex-independent up-regulation in hippocampal nerve growth factor-inducible gene B mRNA expression that does not require CaMKKalpha. Thus, we show that CaMKKalpha has a specific complex role in memory formation in males.

Dual role for TGF-beta1 in apoptosis.

The exposure of cells to TGF-beta1 can trigger a variety of cellular responses including the inhibition of cell growth, migration, differentiation and apoptosis. TGF-beta1-regulated apoptosis is cell type and context-dependent, indeed TGF-beta1 provides signals for both cell survival or apoptosis. The molecular mechanisms underlying the role of TGF-beta1 in apoptosis remains unclear. The proteins that primarily mediate the intracellular signaling of TGF-beta1 are the members of the Smad family. Nevertheless, TGF-beta1 signaling can also cooperate with the death receptor apoptotic pathway (Fas, TNF), with the intracellular modulators of apoptosis JNK and p38 MAP kinases, Akt, NF-kappaB, and with the mitochondrial apoptotic pathway mediated by members of the Bcl-2 family. Moreover, the involvement of TGF-beta1 in the production of oxidative stress and in preventing the inflammatory processes required for the clearance of apoptotic bodies is further evidence of its integration into apoptotic pathways. The interaction and balance between different stimuli provides the basis for the pro- or anti-apoptotic output of TGF-beta1 signaling in a given cell.

Transforming growth factor beta1 overexpression in the nigrostriatal system increases the dopaminergic deficit of MPTP mice.

Idiopathic Parkinson's disease (PD) is characterized by mesencephalic dopaminergic neuron cell death and striatal dopamine (DA) depletion. The factors involved in the pathogenesis of the disease are still unknown. Transforming growth factor beta1 (TGFbeta1) is increased in the striatum of patients with PD. However, the effect of this increase is not known. Here, we show that overexpression of TGFbeta1, by recombinant adenovirus TGFbeta1 gene transfer, in the mesostriatal system of an MPTP mouse model of PD decreased the number of mesencephalic dopaminergic neurons. This effect also involved more extensive DA depletion in the striatum. Striatal enkephalin mRNA levels are augmented, suggesting a decrease in dopaminergic transmission to the postsynaptic target. In the absence of MPTP, TGFbeta1 greatly decreased the number of dopaminergic neurons in the ventral mesencephalon of fully mature mice. These results show that an increase in TGFbeta1 levels aggravate the parkinsonian status of MPTP mice and may therefore be a risk factor for the development of PD.

Inducible cAMP early repressor, an endogenous antagonist of cAMP responsive element-binding protein, evokes neuronal apoptosis in vitro.

Active CREB (cAMP responsive element-binding protein) transcription factor is crucial for neuronal survival. Several members of the CREM/ICER (cAMP responsive element modulator/inducible cAMP early repressor) protein family may act as endogenous CREB antagonists. However, their involvement in a process of programmed cell death remains unexplored. Here we report that ICER may play such a role in neuronal apoptosis because it is upregulated in apoptotic neurons in vitro, and overexpression of ICER, delivered in adenoviral vector, evokes programmed cell death of three different kinds of cultured neurons, namely those derived from hippocampal dentate gyrus, cerebral cortex, and superior cervical ganglion. Reporter gene assay with a promoter containing a CREB-responsive sequence revealed a decrease in both basal and induced CRE-dependent gene expression in neurons overexpressing ICER. Finally, the level of expression of the anti-apoptotic protein Bcl-2, a well known CREB target, was markedly diminished in ICER-treated neurons. We suggest that the naturally occurring CREB functional antagonist ICER may have a specific function in programmed cell death of neurons, probably by silencing the expression of anti-apoptotic genes.