α-synuclein (α-syn) is the main component of intracytoplasmic inclusions deposited in the brains of patients with Parkinson's disease (PD) and certain other neurodegenerative disorders. Recent studies have explored the ability of α-syn to propagate between or across neighboring neurons and supposedly "infect" them with a prion-like mechanism. However, much of this research has used stereotaxic injections of heterologous α-syn fibrils to induce the spreading of inclusions in the rodent brains. Whether α-syn is able to transmit from the host cells to their neighboring cells in vivo is unclear.

Dopamine (DA) release in striatum is functionally segregated across a dorsolateral/ventromedial axis. Interestingly, nigrostriatal DA signaling disruption in Parkinson's disease (PD) preferentially affects the dorsolateral striatum. The relationship between afferent presynaptic calcium transients (PreCaTs) in DA terminals and DA release in dorsolateral (Caudato-Putamen, DLS) and ventromedial (Nucleus Accumbens Shell, VS) striatal subregions was examined by ex vivo real-time dual-recording in conditional transgenic mice expressing the calcium indicator protein GCaMP3. In DLS, minimal increases in cytosolic calcium trigger steep DA release while PreCaTs and DA release in VS both were proportional to the number of pulses in burst stimulation. Co-expressing α-synuclein with the Parkinson's disease (PD)-associated A53T mutation and GCaMP3 in midbrain DA neurons revealed augmented cytosolic steady state and activity-dependent intra-terminal calcium levels preferentially in DLS, as well as hyperactivation and enhanced expression of N-type calcium channels. Thus, unbalanced calcium channel activity is a presynaptic mechanism to consider in the multifaceted pathogenic pathways of progressive neurodegeneration.

Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1-/- mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1+/- mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.

Although the role of hippocampus in memory processing is well assessed, an association of experience-dependent behavioural modifications with hippocampal neuron morphological and biochemical changes deserves further characterisation. Here, we present evidence of dendritic alterations together with rapid accumulation of EphrinB2, a factor known to influence cell plasticity, in pyramidal neurons of the CA1 area of mouse hippocampus, during the formation of recent contextual fear memory. Male C57BL/6N mice exhibited a robust fear response 24h after contextual and cued fear conditioning. At this time and in the absence of the memory test, conditioned mice showed morphological alterations in hippocampal and lateral amygdala neurons. Western blot analysis of extracts from conditioned but not pseudoconditioned or naive mice showed a specific increase in the amount of EphrinB2 in the hippocampus but not the cortex. However, levels of EphA4 receptor, known to interact trans-synaptically with EphrinB2, did not change upon conditioning in extracts from the same structures. Finally, immunohistochemical analysis of the hippocampus and amygdala of conditioned mice showed increased levels of EphrinB2 in pyramidal neurons of the CA1 area, when compared to pseudoconditioned and control mice. Such increase was not observed in other hippocampal areas or the amygdala. These results suggest that rapid accumulation of EphrinB2 in hippocampal CA1 neurons is involved in the behavioural and cellular modifications induced by contextual fear conditioning. A similar mechanism does not appear to occur in lateral amygdala neurons, in spite of the robust behavioural and cellular modifications induced in such structure by cued fear conditioning.

Multiple missense mutations in p150Glued are linked to Perry syndrome (PS), a rare neurodegenerative disease pathologically characterized by loss of nigral dopaminergic (DAergic) neurons. Here we generated p150Glued conditional knockout (cKO) mice by deleting p150Glued in midbrain DAergic neurons. The young cKO mice displayed impaired motor coordination, dystrophic DAergic dendrites, swollen axon terminals, reduced striatal dopamine transporter (DAT), and dysregulated dopamine transmission. The aged cKO mice showed loss of DAergic neurons and axons, somatic accumulation of α-synuclein, and astrogliosis. Further mechanistic studies revealed that p150Glued deficiency in DAergic neurons led to the reorganization of endoplasmic reticulum (ER) in dystrophic dendrites, upregulation of ER tubule-shaping protein reticulon 3, accumulation of DAT in reorganized ERs, dysfunction of COPII-mediated ER export, activation of unfolded protein response, and exacerbation of ER stress-induced cell death. Our findings demonstrate the importance of p150Glued in controlling the structure and function of ER, which is critical for the survival and function of midbrain DAergic neurons in PS.

Alzheimer's disease (AD) is thought to be caused by accumulation of amyloid-β protein (Aβ), which is a cleavage product of amyloid precursor protein (APP). Transgenic mice overexpressing APP have been used to recapitulate amyloid-β pathology. Among them, APP23 and APPswe/PS1deltaE9 (deltaE9) mice are extensively studied. APP23 mice express APP with Swedish mutation and develop amyloid plaques late in their life, while cognitive deficits are observed in young age. In contrast, deltaE9 mice with mutant APP and mutant presenilin-1 develop amyloid plaques early but show typical cognitive deficits in old age. To unveil the reasons for different progressions of cognitive decline in these commonly used mouse models, we analyzed the number and turnover of dendritic spines as important structural correlates for learning and memory. Chronic in vivo two-photon imaging in apical tufts of layer V pyramidal neurons revealed a decreased spine density in 4-5-month-old APP23 mice. In age-matched deltaE9 mice, in contrast, spine loss was only observed on cortical dendrites that were in close proximity to amyloid plaques. In both cases, the reduced spine density was caused by decreased spine formation. Interestingly, the patterns of alterations in spine morphology differed between these two transgenic mouse models. Moreover, in APP23 mice, APP was found to accumulate intracellularly and its content was inversely correlated with the absolute spine density and the relative number of mushroom spines. Collectively, our results suggest that different pathological mechanisms, namely an intracellular accumulation of APP or extracellular amyloid plaques, may lead to spine abnormalities in young adult APP23 and deltaE9 mice, respectively. These distinct features, which may represent very different mechanisms of synaptic failure in AD, have to be taken into consideration when translating results from animal studies to the human disease.

Dopamine replacement with levodopa (L-DOPA) represents the mainstay of Parkinson’s disease (PD) therapy. Nevertheless, this well established therapeutic intervention loses efficacy with the progression of the disease and patients develop invalidating side effects, known in their complex as L-DOPA-induced dyskinesia (LID). Unfortunately, existing therapies fail to prevent LID and very few drugs are available to lessen its severity, thus representing a major clinical problem inPDtreatment. D2-like receptor (D2R) agonists are a powerful clinical option as an alternative to L-DOPA, especially in the early stages of the disease, being associated to a reduced risk of dyskinesia development. D2R agonists also find considerable application in the advanced stages of PD, in conjunction with L-DOPA, which is used in this context at lower dosages, to delay the appearance and the extent of the motor complications. In advanced stages of PD, D2R agonists are often effective in delaying the appearance and the extent of motor complications. Despite the great attention paid to the family of D2R agonists, the main reasons underlying the reduced risk of dyskinesia have not yet been fully characterized. Here we show that the striatal NMDA/AMPAreceptor ratio and theAMPAreceptor subunit composition are altered in experimental parkinsonism in rats. Surprisingly, while L-DOPA fails to restore these critical synaptic alterations, chronic treatment with pramipexole is associated not only with a reduced risk of dyskinesia development but is also able to rebalance, in a dose-dependent fashion, the physiological synaptic parameters, thus providing new insights into the mechanisms of dyskinesia.

Aldehyde dehydrogenase 1 (ALDH1A1)-positive dopaminergic (DA) neurons at the ventral substantia nigra pars compacta (SNpc) preferentially degenerate in Parkinson's disease (PD). Their projection pattern and dopamine release properties, however, remains uncharacterized. Here we show that ALDH1A1-positive axons project predominantly to the rostral two-thirds of dorsal striatum. A portion of these axons converge on a small fraction of striosome compartments restricted to the dorsolateral striatum (DLS), where less dopamine release was measured compared to the adjacent matrix enriched with the ALDH1A1-negative axons. Genetic ablation of Aldh1a1 substantially increases the dopamine release in striosomes, but not in matrix. Additionally, the presence of PD-related human α-synuclein A53T mutant or dopamine transporter (DAT) blockers also differentially affects the dopamine output in striosomes and matrix. Together, these results demonstrate distinct dopamine release characteristics of ALDH1A1-positive DA fibers, supporting a regional specific function of ALDH1A1 in regulating dopamine availability/release in striatum.

In Alzheimer's disease, BACE1 protease initiates the amyloidogenic processing of amyloid precursor protein (APP) that eventually results in synthesis of β-amyloid (Aβ) peptide. Aβ deposition in turn causes accumulation of BACE1 in plaque-associated dystrophic neurites, thereby potentiating progressive Aβ deposition once initiated. Since systemic pharmacological BACE inhibition causes adverse effects in humans, it is important to identify strategies that specifically normalize overt BACE1 activity around plaques. The microtubule-associated protein tau regulates axonal transport of proteins, and tau deletion rescues Aβ-induced transport deficits in vitro. In the current study, long-term in vivo two-photon microscopy and immunohistochemistry were performed in tau-deficient APPPS1 mice. Tau deletion reduced plaque-associated axonal pathology and BACE1 accumulation without affecting physiological BACE1 expression distant from plaques. Thereby, tau deletion effectively decelerated formation of new plaques and reduced plaque compactness. The data revealed that tau reinforces Aβ deposition, presumably by contributing to accumulation of BACE1 in plaque-associated dystrophies. Targeting tau-dependent mechanisms could become a suitable strategy to specifically reduce overt BACE1 activity around plaques, thereby avoiding adverse effects of systemic BACE inhibition.

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is a promising drug target for the treatment of Alzheimer's disease. Prolonged BACE1 inhibition interferes with structural and functional synaptic plasticity in mice, most likely by altering the metabolism of BACE1 substrates. Seizure protein 6 (SEZ6) is predominantly cleaved by BACE1, and Sez6 knockout mice share some phenotypes with BACE1 inhibitor-treated mice. We investigated whether SEZ6 is involved in BACE1 inhibition-induced structural and functional synaptic alterations.

Misfolded α-synuclein spreads along anatomically connected areas through the brain, prompting progressive neurodegeneration of the nigrostriatal pathway in Parkinson's disease. To investigate the impact of early stage seeding and spreading of misfolded α-synuclein along with the nigrostriatal pathway, we studied the pathophysiologic effect induced by a single acute α-synuclein preformed fibrils (PFFs) inoculation into the midbrain. Further, to model the progressive vulnerability that characterizes the dopamine (DA) neuron life span, we used two cohorts of mice with different ages: 2-month-old (young) and 5-month-old (adult) mice. Two months after α-synuclein PFFs injection, we found that striatal DA release decreased exclusively in adult mice. Adult DA neurons showed an increased level of pathology spreading along with the nigrostriatal pathway accompanied with a lower volume of α-synuclein deposition in the midbrain, impaired neurotransmission, rigid DA terminal composition, and less microglial reactivity compared with young neurons. Notably, preserved DA release and increased microglial coverage in the PFFs-seeded hemisphere coexist with decreased large-sized terminal density in young DA neurons. This suggests the presence of a targeted pruning mechanism that limits the detrimental effect of α-synuclein early spreading. This study suggests that the impact of the pathophysiology caused by misfolded α-synuclein spreading along the nigrostriatal pathway depends on the age of the DA network, reducing striatal DA release specifically in adult mice.

Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). We found that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIβ (PKARIIβ). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged Lrrk2(-/-) mice after treatment with a Drd1 agonist. Notably, a Parkinson's disease-related Lrrk2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIβ also induced excessive PKA activity in the SPNs. Our findings reveal a previously unknown regulatory role for LRRK2 in PKA signaling and suggest a pathogenic mechanism of SPN dysfunction in Parkinson's disease.

Dynamic synapses facilitate activity-dependent remodeling of neural circuits, thereby providing the structural substrate for adaptive behaviors. However, the mechanisms governing dynamic synapses in adult brain are still largely unknown. Here, we demonstrate that in the cortex of adult amyloid precursor protein knockout (APP-KO) mice, spine formation and elimination were both reduced while overall spine density remained unaltered. When housed under environmental enrichment, APP-KO mice failed to respond with an increase in spine density. Spine morphology was also altered in the absence of APP The underlying mechanism of these spine abnormalities in APP-KO mice was ascribed to an impairment in D-serine homeostasis. Extracellular D-serine concentration was significantly reduced in APP-KO mice, coupled with an increase of total D-serine. Strikingly, chronic treatment with exogenous D-serine normalized D-serine homeostasis and restored the deficits of spine dynamics, adaptive plasticity, and morphology in APP-KO mice. The cognitive deficit observed in APP-KO mice was also rescued by D-serine treatment. These data suggest that APP regulates homeostasis of D-serine, thereby maintaining the constitutive and adaptive plasticity of dendritic spines in adult brain.

Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

Although the role of noxious α-synuclein (α-SYN) in the degeneration of midbrain dopaminergic neurons and associated motor deficits of Parkinson's disease is recognized, its impact on non-motor brain circuits and related symptoms remains elusive. Through combining in vivo two-photon imaging with time-coded labelling of neurons in the olfactory bulb of A30P α-SYN transgenic mice, we show impaired growth and branching of dendrites of adult-born granule cells (GCs), with reduced gain and plasticity of dendritic spines. The spine impairments are especially pronounced during the critical phase of integration of new neurons into existing circuits. Functionally, retarded dendritic expansion translates into reduced electrical capacitance with enhanced intrinsic excitability and responsiveness of GCs to depolarizing inputs, while the spine loss correlates with decreased frequency of AMPA-mediated miniature EPSCs. Changes described here are expected to interfere with the functional integration and survival of new GCs into bulbar networks, contributing towards olfactory deficits and related behavioural impairments.

A major neuropathological hallmark of Alzheimer's disease is the deposition of amyloid plaques in the brains of affected individuals. Amyloid plaques mainly consist of fibrillar β-amyloid, which is a cleavage product of the amyloid precursor protein. The amyloid-cascade-hypothesis postulates Aβ accumulation as the central event in initiating a toxic cascade leading to Alzheimer's disease pathology and, ultimately, loss of cognitive function. We studied the kinetics of β-amyloid deposition in Tg2576 mice, which overexpress human amyloid precursor protein with the Swedish mutation. Utilizing long-term two-photon imaging we were able to observe the entire kinetics of plaque growth in vivo. Essentially, we observed that plaque growth follows a sigmoid-shaped curve comprising a cubic growth phase, followed by saturation. In contrast, plaque density kinetics exhibited an asymptotic progression. Taking into account the fact that a critical concentration of Aβ is required to seed new plaques, we can propose the following kinetic model of β-amyloid deposition in vivo. In the early cubic phase, plaque growth is not limited by Aβ concentration and plaque density increases very fast. During the transition phase, plaque density stabilizes whereas plaque volume increases strongly reflecting a robust growth of the plaques. In the late asymptotic phase, Aβ peptide production becomes rate-limiting for plaque growth. In conclusion, the present study offers a direct link between in vitro and in vivo studies facilitating the translation of Aβ-lowering strategies from laboratory models to patients.

BACE1 is the rate-limiting protease in the production of synaptotoxic β-amyloid (Aβ) species and hence one of the prime drug targets for potential therapy of Alzheimer's disease (AD). However, so far pharmacological BACE1 inhibition failed to rescue the cognitive decline in mild-to-moderate AD patients, which indicates that treatment at the symptomatic stage might be too late. In the current study, chronic in vivo two-photon microscopy was performed in a transgenic AD model to monitor the impact of pharmacological BACE1 inhibition on early β-amyloid pathology. The longitudinal approach allowed to assess the kinetics of individual plaques and associated presynaptic pathology, before and throughout treatment. BACE1 inhibition could not halt but slow down progressive β-amyloid deposition and associated synaptic pathology. Notably, the data revealed that the initial process of plaque formation, rather than the subsequent phase of gradual plaque growth, is most sensitive to BACE1 inhibition. This finding of particular susceptibility of plaque formation has profound implications to achieve optimal therapeutic efficacy for the prospective treatment of AD.

All clinical BACE1-inhibitor trials for the treatment of Alzheimer's Disease (AD) have failed due to insufficient efficacy or side effects like worsening of cognitive symptoms. However, the scientific evidence to date suggests that BACE1-inhibition could be an effective preventative measure if applied prior to the accumulation of amyloid-beta (Aβ)-peptide and resultant impairment of synaptic function. Preclinical studies have associated BACE1-inhibition-induced cognitive deficits with decreased dendritic spine density. Therefore, we investigated dose-dependent effects of BACE1-inhibition on hippocampal dendritic spine dynamics in an APP knock-in mouse line for the first time. We conducted in vivo two-photon microscopy in the stratum oriens layer of hippocampal CA1 neurons in 3.5-month-old App NL-G-F GFP-M mice over 6 weeks to monitor the effect of potential preventive treatment with a high and low dose of the BACE1-inhibitor NB-360 on dendritic spine dynamics. Structural spine plasticity was severely impaired in untreated App NL-G-F GFP-M mice, although spines were not yet showing signs of degeneration. Prolonged high-dose BACE1-inhibition significantly enhanced spine formation, improving spine dynamics in the AD mouse model. We conclude that in an early AD stage characterized by low Aβ-accumulation and no irreversible spine loss, BACE1-inhibition could hold the progressive synapse loss and cognitive decline by improving structural spine dynamics.

Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood-brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.

We identified a rare undifferentiated cell population that is intermingled with the Bergmann glia of the adult murine cerebellar cortex, expresses the stem cell markers Sox2 and Nestin, and lacks markers of glial or neuronal differentiation. Interestingly, such Sox2+ S100- cells of the adult cerebellum expanded after adequate physiological stimuli in mice (exercise), and Sox2+ precursors acquired positivity for the neuronal marker NeuN over time and integrated into cellular networks. In human patients, SOX2+ S100- cells similarly increased in number after relevant pathological insults (infarcts), suggesting a similar expansion of cells that lack terminal glial differentiation.