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Fatty acid elongase ELOVL5 is part of a protein family of multipass transmembrane proteins that reside in the endoplasmic reticulum where they regulate long-chain fatty acid elongation. A missense variant (c.689G > T p.Gly230Val) in ELOVL5 causes Spinocerebellar Ataxia subtype 38 (SCA38), a neurodegenerative disorder characterized by autosomal dominant inheritance, cerebellar Purkinje cell demise and adult-onset ataxia. Having previously showed aberrant accumulation of p.G230V in the Golgi complex, here we further investigated the pathogenic mechanisms triggered by p.G230V, integrating functional studies with bioinformatic analyses of protein sequence and structure. Biochemical analysis showed that p.G230V enzymatic activity was normal. In contrast, SCA38-derived fibroblasts showed reduced expression of ELOVL5, Golgi complex enlargement and increased proteasomal degradation with respect to controls. By heterologous overexpression, p.G230V was significantly more active than wild-type ELOVL5 in triggering the unfolded protein response and in decreasing viability in mouse cortical neurons. By homology modelling, we generated native and p.G230V protein structures whose superposition revealed a shift in Loop 6 in p.G230V that altered a highly conserved intramolecular disulfide bond. The conformation of this bond, connecting Loop 2 and Loop 6, appears to be elongase-specific. Alteration of this intramolecular interaction was also observed when comparing wild-type ELOVL4 and the p.W246G variant which causes SCA34. We demonstrate by sequence and structure analyses that ELOVL5 p.G230V and ELOVL4 p.W246G are position-equivalent missense variants. We conclude that SCA38 is a conformational disease and propose combined loss of function by mislocalization and gain of toxic function by ER/Golgi stress as early events in SCA38 pathogenesis.

The transcription factor NURR1 is essential to the generation and maintenance of midbrain dopaminergic (mDA) neurons and its deregulation is involved in the development of dopamine (DA)-associated brain disorders, such as Parkinson's disease (PD). The old male NURR1 heterozygous knockout (NURR1-KO) mouse has been proposed as a model of PD due to its altered motor performance that was, however, not confirmed in a subsequent study. Based on these controversial results, we explored the effects of the NURR1 deficiency on locomotor activity, motor coordination, brain and plasma DA levels, blood pressure and heart rate of old mice, also focusing on the potential effect of sex. As a probable consequence of the role of NURR1 in DA pathway, we observed that the old NURR1-KO mouse is characterized by motor impairment, and increased brain DA level and heart rate, independently from sex. However, we also observed an alteration in spontaneous locomotor activity that only affects males. In conclusion, NURR1 deficiency triggers sex- and age-specific alterations of behavioral responses, of DA levels and cardiovascular abnormalities. Further studies in simplified systems will be necessary to dissect the mechanism underlying these observations.

Elovl5 elongates fatty acids with 18 carbon atoms and in cooperation with other enzymes guarantees the normal levels of very long‐chain fatty acids, which are necessary for a proper membrane structure. Action potential conduction along myelinated axons depends on structural integrity of myelin, which is maintained by a correct amount of fatty acids and a proper interaction between fatty acids and myelin proteins. We hypothesized that in Elovl5 −/− mice, the lack of elongation of Elovl5 substrates might cause alterations of myelin structure. The analysis of myelin ultrastructure showed an enlarged periodicity with reduced G‐ratio across all axonal diameters. We hypothesized that the structural alteration of myelin might affect the conduction of action potentials. The sciatic nerve conduction velocity was significantly reduced without change in the amplitude of the nerve compound potential, suggesting a myelin defect without a concomitant axonal degeneration. Since Elovl5 is important in attaining normal amounts of polyunsaturated fatty acids, which are the principal component of myelin, we performed a lipidomic analysis of peripheral nerves of Elovl5‐deficient mice. The results revealed an unbalance, with reduction of fatty acids longer than 18 carbon atoms relative to shorter ones. In addition, the ratio of saturated to unsaturated fatty acids was strongly increased. These findings point out the essential role of Elovl5 in the peripheral nervous system in supporting the normal structure of myelin, which is the key element for a proper conduction of electrical signals along myelinated nerves., MAIN POINTS Consequences on peripheral nerves of Elovl5 deletion:Unbalance between saturated versus unsaturated fatty acids in membrane phospholipids.Loss of myelin compactnessSlower action potential conduction

G-protein-coupled inwardly rectifying potassium (GIRK) channels contribute to the resting membrane potential of many neurons and play an important role in controlling neuronal excitability. Although previous studies have revealed a high expression of GIRK subunits in the cerebellum, their functional role has never been clearly described. Using patch-clamp recordings in mice cerebellar slices, we examined the properties of the GIRK currents in Purkinje cells (PCs) and investigated the effects of a selective agonist of GIRK1-containing channels, ML297 (ML), on PC firing and synaptic plasticity. We demonstrated that GIRK channel activation decreases the PC excitability by inhibiting both sodium and calcium spikes and, in addition, modulates the complex spike response evoked by climbing fiber stimulation. Our results indicate that GIRK channels have also a marked effect on synaptic plasticity of the parallel fiber-PC synapse, as the application of ML297 increased the expression of LTP while preventing LTD. We, therefore, propose that the recruitment of GIRK channels represents a crucial mechanism by which neuromodulators can control synaptic strength and membrane conductance for proper refinement of the neural network involved in memory storage and higher cognitive functions.

Convergent lines of evidence have recently highlighted β3-adrenoreceptors (ARs) as a potentially critical target in the regulation of nervous and behavioral functions, including memory consolidation, anxiety, and depression. Nevertheless, the role of β3-ARs in the cerebellum has been never investigated. To address this issue, we first examined the effects of pharmacological manipulation of β3-ARs on motor learning in mice. We found that blockade of β3-ARs by SR 59230A impaired the acquisition of the rotarod task with no effect on general locomotion. Since the parallel fiber-Purkinje cell (PF-PC) synapse is considered to be the main cerebellar locus of motor learning, we assessed β3-AR modulatory action on this synapse as well as its expression in cerebellar slices. We demonstrate, for the first time, a strong expression of β3-ARs on Purkinje cell soma and dendrites. In addition, whole-cell patch-clamp recordings revealed that bath application of β3-AR agonist CL316,243 depressed the PF-PC excitatory postsynaptic currents via a postsynaptic mechanism mediated by the PI3K signaling pathway. Application of CL316,243 also interfered with the expression of PF long-term potentiation, whereas SR 59230A prevented the induction of LTD at PF-PC synapse. These results underline the critical role of β3-AR on cerebellar synaptic transmission and plasticity and provide a new mechanism for adrenergic modulation of motor learning.

The 5-HT7 receptor (5-HT7R) mediates important physiological effects of serotonin, such as memory and emotion, and is emerging as a therapeutic target for the treatment of cognitive disorders and depression. Although previous studies have revealed an expression of 5-HT7R in cerebellum, particularly at Purkinje cells, its functional role and signaling mechanisms have never been described. Using patch-clamp recordings in cerebellar slices of adult mice, we investigated the effects of a selective 5-HT7R agonist, LP-211, on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that 5-HT7R activation induces long-term depression of parallel fiber-Purkinje cell synapse via a postsynaptic mechanism that involves the PKC-MAPK signaling pathway. Moreover, a 5-HT7R antagonist abolished the expression of PF-LTD, produced by pairing parallel fiber stimulation with Purkinje cell depolarization; whereas, application of a 5-HT7R agonist impaired LTP induced by 1 Hz parallel fiber stimulation. Our results indicate for the first time that 5-HT7R exerts a fine regulation of cerebellar bidirectional synaptic plasticity that might be involved in cognitive processes and neuropsychiatric disorders involving the cerebellum.

Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but signs of ataxia were detectable by beam test at 18 months. Cerebellar pathology was negative; electrophysiological analysis showed increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls, although not statistically significant. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was also altered, in line with greatly reduced expression of Opa1 fusogenic protein L-isoforms. The mitochondrial alterations observed in MEFs were also detected in cerebella of 18-month-old heterozygous mutants, suggesting they may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.

Sexual disturbances, and aggressivity are a major social problem. However, the molecular mechanisms involved in the control of these behaviors are largely unknown. FGF14, which is an intracellular protein controlling neuronal excitability and synaptic transmission, has been implied in neurologic and psychiatric disorders. Mice with Fgf14 deletion show blunted responses to drugs of abuse. By behavioral tests we show that male Fgf14 knockout mice have a marked reduction of several behaviors including aggressivity and sexual behavior. Other behaviors driven by spontaneous initiative like burying novel objects and spontaneous digging and climbing are also reduced in Fgf14 knockout mice. These deficits cannot be attributed to a generalized decrease of activity levels, because in the open field test Fgf14 knockout mice have the same spontaneous locomotion as wild types and increased rearing. Our results show that Fgf14 is important to preserve a set of behaviors and suggest that fine tuning of neuronal function by Fgf14 is an important mechanism of control for such behaviors.

The formation of the complex cerebellar cortical circuits follows different phases, with initial synaptogenesis and subsequent processes of refinement guided by a variety of mechanisms. The regularity of the cellular and synaptic organization of the cerebellar cortex allowed detailed studies of the structural plasticity mechanisms underlying the formation of new synapses and retraction of redundant ones. For the attainment of the monoinnervation of the Purkinje cell by a single climbing fiber, several signals are involved, including electrical activity, contact signals, homosynaptic and heterosynaptic interaction, calcium transients, postsynaptic receptors, and transduction pathways. An important role in this developmental program is played by serotonergic projections that, acting on temporally and spatially regulated postsynaptic receptors, induce and modulate the phases of synaptic formation and maturation. In the adult cerebellar cortex, many developmental mechanisms persist but play different roles, such as supporting synaptic plasticity during learning and formation of cerebellar memory traces. A dysfunction at any stage of this process can lead to disorders of cerebellar origin, which include autism spectrum disorders but are not limited to motor deficits. Recent evidence in animal models links impairment of Purkinje cell function with autism-like symptoms including sociability deficits, stereotyped movements, and interspecific communication by vocalization.

Mice deficient for the transcription factor Ebf2 lose a subset of Purkinje cells during development and have a hypotrophic cerebellar cortex. Related motor symptoms and the function of Purkinje cells surviving in the adult have not been studied so far. Ebf2 null mice presented locomotor impairment and a deficiency of motor coordination and motor learning. Ebf2 null Purkinje cells of the anterior lobe, relative to wild-type controls, were patch-clamp recorded in acutely prepared slices. While immature Purkinje cells (10-20 postnatal days) of Ebf2 null mice showed no significant difference relative to wild-types, in the adult they featured a higher input resistance, increased anomalous rectification, decreased first spike latency, higher initial firing frequency, lower voltage threshold and reduced afterhyperpolarizations and post-burst hyperpolarizations. These parameters indicate a difference in the response to both hyperpolarizing and depolarizing stimuli, corresponding to an altered cerebellar cortical output signaling. In contrast, adult climbing fibers attained a normal monoinnervation pattern and the parallel fiber-Purkinje cell synapse showed evoked postsynaptic currents and paired-pulse facilitation functionally indistinguishable from wild-type PCs. These results suggest that the motor deficits exhibited by Ebf2 null mice could be due, at least in part, to an impairment of the firing properties of surviving Purkinje cells. These findings indicate that Ebf2 is important for the development and maintenance of normal Purkinje cell discharge properties. © 2012 Elsevier Inc.

The parallel fiber-Purkinje cell synapse represents the point of maximal signal divergence in the cerebellar cortex with an estimated number of about 60 billion synaptic contacts in the rat and 100,000 billions in humans. At the same time, the Purkinje cell dendritic tree is a site of remarkable convergence of more than 100,000 parallel fiber synapses. Parallel fibers activity generates fast postsynaptic currents via AMPA receptors, and slower signals, mediated by mGlu1 receptors, resulting in Purkinje cell depolarization accompanied by sharp calcium elevation within dendritic regions. Long-term depression and long-term potentiation have been widely described for the parallel fiber-Purkinje cell synapse and have been proposed as mechanisms for motor learning. The mechanisms of induction for LTP and LTD involve different signaling mechanisms within the presynaptic terminal and/or at the postsynaptic site, promoting enduring modification in the neurotransmitter release and change in responsiveness to the neurotransmitter. The parallel fiber-Purkinje cell synapse is finely modulated by several neurotransmitters, including serotonin, noradrenaline, and acetylcholine. The ability of these neuromodulators to gate LTP and LTD at the parallel fiber-Purkinje cell synapse could, at least in part, explain their effect on cerebellar-dependent learning and memory paradigms. Overall, these findings have important implications for understanding the cerebellar involvement in a series of pathological conditions, ranging from ataxia to autism. For example, parallel fiber-Purkinje cell synapse dysfunctions have been identified in several murine models of spinocerebellar ataxia (SCA) types 1, 3, 5 and 27. In some cases, the defect is specific for the AMPA receptor signaling (SCA27), while in others the mGlu1 pathway is affected (SCA1, 3, 5). Interestingly, the parallel fiber-Purkinje cell synapse has been shown to be hyper-functional in a mutant mouse model of autism spectrum disorder, with a selective deletion of Pten in Purkinje cells. However, the full range of methodological approaches, that allowed the discovery of the physiological principles of PF-PC synapse function, has not yet been completely exploited to investigate the pathophysiological mechanisms of diseases involving the cerebellum. We, therefore, propose to extend the spectrum of experimental investigations to tackle this problem.

The signals arriving to Purkinje cells via parallel fibers are essential for all tasks in which the cerebellum is involved, including motor control, learning new motor skills and calibration of reflexes. Since learning also requires the activation of adrenergic receptors, we investigated the effects of adrenergic receptor agonists on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that noradrenaline serves as an endogenous ligand for both α1-and α2-adrenergic receptors to produce synaptic depression between parallel fibers and Purkinje cells. On the contrary, PF-EPSCs were potentiated by the β-adrenergic receptor agonist isoproterenol. This short-term potentiation was postsynaptically expressed, required protein kinase A, and was mimicked by the β2-adrenoceptor agonist clenbuterol, suggesting that the β2-adrenoceptors mediate the noradrenergic facilitation of synaptic transmission between parallel fibers and Purkinje cells. Moreover, β-adrenoceptor activation lowered the threshold for cerebellar long-term potentiation induced by 1 Hz parallel fiber stimulation. The presence of both α and β adrenergic receptors on Purkinje cells suggests the existence of bidirectional mechanisms of regulation allowing the noradrenergic afferents to refine the signals arriving to Purkinje cells at particular arousal states or during learning.

Cognitive processing is highly dependent on the functional integrity of gamma-amino-butyric acid (GABA) interneurons in the brain. These cells regulate excitability and synaptic plasticity of principal neurons balancing the excitatory/inhibitory tone of cortical networks. Reduced function of parvalbumin (PV) interneurons and disruption of GABAergic synapses in the cortical circuitry result in desynchronized network activity associated with cognitive impairment across many psychiatric disorders, including schizophrenia. However, the mechanisms underlying these complex phenotypes are still poorly understood. Here we show that in animal models, genetic deletion of fibroblast growth factor 14 (Fgf14), a regulator of neuronal excitability and synaptic transmission, leads to loss of PV interneurons in the CA1 hippocampal region, a critical area for cognitive function. Strikingly, this cellular phenotype associates with decreased expression of glutamic acid decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) and also coincides with disrupted CA1 inhibitory circuitry, reduced in vivo gamma frequency oscillations and impaired working memory. Bioinformatics analysis of schizophrenia transcriptomics revealed functional co-clustering of FGF14 and genes enriched within the GABAergic pathway along with correlatively decreased expression of FGF14, PVALB, GAD67 and VGAT in the disease context. These results indicate that Fgf14−/− mice recapitulate salient molecular, cellular, functional and behavioral features associated with human cognitive impairment, and FGF14 loss of function might be associated with the biology of complex brain disorders such as schizophrenia.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impaired social interaction, isolated areas of interest, and insistence on sameness. Mutations in Phosphatase and tensin homolog missing on chromosome 10 (PTEN) have been reported in individuals with ASDs. Recent evidence highlights a crucial role of the cerebellum in the etiopathogenesis of ASDs. In the present study we analyzed the specific contribution of cerebellar Purkinje cell (PC) PTEN loss to these disorders. Using the Cre-loxP recombination system, we generated conditional knockout mice in which PTEN inactivation was induced specifically in PCs. We investigated PC morphology and physiology as well as sociability, repetitive behavior, motor learning, and cognitive inflexibility of adult PC PTEN-mutant mice. Loss of PTEN in PCs results in autistic-like traits, including impaired sociability, repetitive behavior and deficits in motor learning. Mutant PCs appear hypertrophic and show structural abnormalities in dendrites and axons, decreased excitability, disrupted parallel fiber and climbing fiber synapses and late-onset cell death. Our results unveil new roles of PTEN in PC function and provide the first evidence of a link between the loss of PTEN in PCs and the genesis of ASD-like traits.

Purpose. Neutrophil-dominated airway inflammation is a key feature of progressive lung damage in cystic fibrosis (CF). Thus, reducing airway inflammation is a major goal to prevent lung damage in CF. However, current anti-inflammatory drugs have shown several limits. PI3Kγplays a pivotal role in leukocyte recruitment and activation; in the present study we determined the effects of genetic deletion and pharmacologic inhibition of PI3Kγon airway inflammation and structural lung damage in a mouse model of CF lung disease.Methods.βENaC overexpressing mice (βENaC-Tg) were backcrossed with PI3Kγ-deficient (PI3KγKO) mice. Tissue damage was assessed by histology and morphometry and inflammatory cell number was evaluated in bronchoalveolar lavage fluid (BALF). Furthermore, we assessed the effect of a specific PI3Kγinhibitor (AS-605240) on inflammatory cell number in BALF.Results. Genetic deletion of PI3Kγdecreased neutrophil numbers in BALF ofPI3KγKO/βENaC-Tg mice, and this was associated with reduced emphysematous changes. Treatment with the PI3Kγinhibitor AS-605240 decreased the number of neutrophils in BALF ofβENaC-Tg mice, reproducing the effect observed with genetic deletion of the enzyme.Conclusions. These results demonstrate the biological efficacy of both genetic deletion and pharmacological inhibition of PI3Kγin reducing chronic neutrophilic inflammation in CF-like lung diseasein vivo.

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.

Enriched environment exposure improves several aspects of cognitive performance in Alzheimer's disease patients and in animal models and, although the role of amyloid plaques is questionable, several studies also assessed their response to enriched environment, with contrasting results. Here we report that rearing APP(Swe)/PS1(L166P) mice in an enriched environment since birth rescued the spatial memory impairment otherwise present at 6 months of age. At the same time, the exposure to the enriched environment caused a transient acceleration of plaque formation, while there was no effect on intracellular staining with the 6E10 antibody, which recognizes β-amyloid, full length amyloid precursor protein and its C-terminal fragments. The anticipation of plaque formation required exposure during early development, suggesting an action within critical periods for circuits formation. On the other hand, chronic neuronal activity suppression by tetrodotoxin decreased the number of plaques without affecting intracellular amyloid. These results indicate that enriched environment exposure since early life has a protective effect on cognitive deterioration although transiently accelerates amyloid deposition. In addition, the effects of the enriched environment might be due to increased neuronal activity, because plaques were reduced by suppression of electrical signaling by tetrodotoxin.

In neurons, voltage-dependent Kv3 potassium channels are essential for the generation of action potentials at high frequency. A dysregulation of the Kv3.1 and Kv3.4 channel subunits has been suggested to contribute to neuronal and glial alterations in Alzheimer's disease, but a quantitative evaluation of these subunits in a mouse model of the pathology is still lacking. We analysed the profile of expression of the four Kv3 subunits by quantitative reverse transcription PCR and Western blot in the whole mouse brain and in dissected brain regions (olfactory bulb, septum, neocortex, hippocampus, brainstem and cerebellum) from 14 days after conception to 18 months after birth. In addition, we measured the levels of Kv3.1 and Kv3.4 messenger RNAs (mRNAs) and proteins in neocortex and hippocampus of APPPS1 mice, a transgenic model of Alzheimer's disease. Although all Kv3 transcripts were significantly expressed in embryonic age in whole brain extracts, only Kv3.1, Kv3.2 and Kv3.4 subunit proteins were present, suggesting a novel role for Kv3 channels at this developmental stage. With the exception of Kv3.4, during postnatal development, Kv3 transcripts and proteins showed a progressive increase in expression and reached an asymptote in adulthood, suggesting that the increase in Kv3 expression during development might contribute to the maturation of the electrical activity of neurons. During aging, Kv3 expression was rather stable. In contrast, in the neocortex of aged APPPS1 mice, Kv3.1 mRNA and protein levels were significantly lower compared to wild type, suggesting that a decrease in Kv3 currents could play a role in the cognitive symptoms of Alzheimer's disease.

Background The m-AAA (A TPases A ssociated with a variety of cellular A ctivities) is an evolutionary conserved metalloprotease complex located in the internal mitochondrial membrane. In the mouse, it is a hetero-oligomer variably formed by the Spg7, Afg3l1, and Afg3l2 encoded proteins, or a homo-oligomer formed by either Afg3l1 or Afg3l2. In humans, AFG3L2 and SPG7 genes are conserved, whereas AFG3L1 became a pseudogene. Both AFG3L2 and SPG7 are involved in a neurodegenerative disease, namely the autosomal dominant spinocerebellar ataxia SCA28 and a recessive form of spastic paraplegia, respectively. Results Using quantitative RT-PCR, we measured the expression levels of Spg7, Afg3l1, and Afg3l2 in the mouse brain. In all regions Afg3l2 is the most abundant transcript, followed by Spg7, and Afg3l1, with a ratio of approximately 5:3:1 in whole-brain mRNA. Using in-situ hybridization, we showed that Spg7, Afg3l1 and Afg3l2 have a similar cellular pattern of expression, with high levels in mitral cells, Purkinje cells, deep cerebellar nuclei cells, neocortical and hippocampal pyramidal neurons, and brainstem motor neurons. However, in some neuronal types, differences in the level of expression of these genes were present, suggesting distinct degrees of contribution of their proteins. Conclusions Neurons involved in SCA28 and hereditary spastic paraplegia display high levels of expression, but similar or even higher expression is also present in other types of neurons, not involved in these diseases, suggesting that the selective cell sensitivity should be attributed to other, still unknown, mechanisms.

Since a growing number of studies based on the real-time reverse transcriptase polymerase chain reaction (RT-PCR) continue to be published in order to highlight genes specifically involved in brain development, maturation, and function, the identification of reference genes suitable for this kind of experiments is now an urgent need in the neuroscience field. The aim of this work was to verify the suitability of some very common housekeeping genes (such as Gapdh, 18s, and B2m) and of some relatively new control genes (such as Pgk1, Tfrc, and Gusb) during mouse brain maturation. We tested the candidate reference genes in mouse whole brain, cerebellum, brain stem, hippocampus, medial septum, frontal neocortex, and olfactory bulb. Moreover, we reported the first complete study of Pgk1 expression throughout the development and the aging of mouse brain. Although no tested gene showed to be the optimal reference for all mouse brain regions, in general, the new housekeeping genes were highly stable in most of the analyzed regions. Above all, with few exceptions, Pgk1 showed to be a reliable control for the analyzed mouse brain regions during development, maturation, and aging.

In Alzheimer's disease (AD), the severity of cognitive symptoms is better correlated with the levels of soluble amyloid-beta (Aβ) rather than with the deposition of fibrillar Aβ in amyloid plaques. In APP/PS1 mice, a murine model of AD, at 8 months of age the cerebellum is devoid of fibrillar Aβ, but dosage of soluble Aβ(1-42), the form which is more prone to aggregation, showed higher levels in this structure than in the forebrain. Aim of this study was to investigate the alterations of intrinsic membrane properties and of synaptic inputs in Purkinje cells (PCs) of the cerebellum, where only soluble Aβ is present. PCs were recorded by whole-cell patch-clamp in cerebellar slices from wild-type and APP/PS1 mice. In APP/PS1 PCs, evoked action potential discharge showed enhanced frequency adaptation and larger afterhyperpolarizations, indicating a reduction of the intrinsic membrane excitability. In the miniature GABAergic postsynaptic currents, the largest events were absent in APP/PS1 mice and the interspike intervals distribution was shifted to the left, but the mean amplitude and frequency were normal. The ryanodine-sensitive multivescicular release was not altered and the postsynaptic responsiveness to a GABA(A) agonist was intact. Climbing fiber postsynaptic currents were normal but their short-term plasticity was reduced in a time window of 100-800 ms. Parallel fiber postsynaptic currents and their short-term plasticity were normal. These results indicate that, in the cerebellar cortex, chronically elevated levels of soluble Aβ(1-42) are associated with alterations of the intrinsic excitability of PCs and with alterations of the release of GABA from interneurons and of glutamate from climbing fibers, while the release of glutamate from parallel fibers and all postsynaptic mechanisms are preserved. Thus, soluble Aβ(1-42) causes, in PCs, multiple functional alterations, including an impairment of intrinsic membrane properties and synapse-specific deficits, with differential consequences even in different subtypes of glutamatergic synapses.