Inhibition of Endocytosis of Clathrin-Mediated Angiotensin II Receptor Type 1 in Podocytes Augments Glomerular Injury.

BACKGROUND: Inhibition of the renin-angiotensin system remains a cornerstone in reducing proteinuria and progression of kidney failure, effects believed to be the result of reduction in BP and glomerular hyperfiltration. However, studies have yielded conflicting results on whether podocyte-specific angiotensin II (AngII) signaling directly induces podocyte injury. Previous research has found that after AngII stimulation, ß-arrestin-bound angiotensin II receptor type 1 (AT1R) is internalized in a clathrin- and dynamin-dependent manner, and that Dynamin1 and Dynamin2 double-knockout mice exhibit impaired clathrin-mediated endocytosis. METHODS: We used podocyte-specific Dyn double-knockout mice to examine AngII-stimulated AT1R internalization and signaling in primary podocytes and controls. We also examined the in vivo effect of AngII in these double-knockout mice through renin-angiotensin system blockers and through deletion of Agtr1a (which encodes the predominant AT1R isoform expressed in kidney, AT1aR). We tested calcium influx, Rac1 activation, and lamellipodial extension in control and primary podocytes of Dnm double-knockout mice treated with AngII. RESULTS: We confirmed augmented AngII-stimulated AT1R signaling in primary Dnm double-knockout podocytes resulting from arrest of clathrin-coated pit turnover. Genetic ablation of podocyte Agtr1a in Dnm double-knockout mice demonstrated improved albuminuria and kidney function compared with the double-knockout mice. Isolation of podocytes from Dnm double-knockout mice revealed abnormal membrane dynamics, with increased Rac1 activation and lamellipodial extension, which was attenuated in Dnm double-knockout podocytes lacking AT1aR. CONCLUSIONS: Our results indicate that inhibiting aberrant podocyte-associated AT1aR signaling pathways has a protective effect in maintaining the integrity of the glomerular filtration barrier.

Dynamin 1- and 3-Mediated Endocytosis Is Essential for the Development of a Large Central Synapse In Vivo.

UNLABELLED: Dynamin is a large GTPase crucial for endocytosis and sustained neurotransmission, but its role in synapse development in the mammalian brain has received little attention. We addressed this question using the calyx of Held (CH), a large nerve terminal in the auditory brainstem in mice. Tissue-specific ablation of different dynamin isoforms bypasses the early lethality of conventional knock-outs and allows us to examine CH development in a native brain circuit. Individual gene deletion of dynamin 1, a primary dynamin isoform in neurons, as well as dynamin 2 and 3, did not affect CH development. However, combined tissue-specific knock-out of both dynamin 1 and 3 (cDKO) severely impaired CH formation and growth during the first postnatal week, and the phenotypes were exacerbated by further additive conditional knock-out of dynamin 2. The developmental defect of CH in cDKO first became evident on postnatal day 3 (P3), a time point when CH forms and grows abruptly. This is followed by a progressive loss of postsynaptic neurons and increased glial infiltration late in development. However, early CH synaptogenesis before protocalyx formation was not altered in cDKO. Functional maturation of synaptic transmission in the medial nucleus of the trapezoid body in cDKO was impeded during development and accompanied by an increase in the membrane excitability of medial nucleus of the trapezoid body neurons. This study provides compelling genetic evidence that CH formation requires dynamin 1- and 3-mediated endocytosis in vivo, indicating a critical role of dynamin in synaptic development, maturation, and subsequent maintenance in the mammalian brain. SIGNIFICANCE STATEMENT: Synaptic development has been increasingly implicated in numerous brain disorders. Dynamin plays a crucial role in clathrin-mediated endocytosis and synaptic transmission at nerve terminals, but its potential role in synaptic development in the native brain circuitry is unclear. Using the calyx of Held, a giant nerve terminal in the mouse brainstem, we evaluated the role of dynamin in this process by using tissue-specific knock-out (KO) of three different dynamin isoforms (dynamin 1, 2, and 3) individually and in combination. Our data demonstrated that dynamin is required for the formation, functional maturation, and subsequent survival of the calyx of Held. This study highlights the important role of dynamin-mediated endocytosis in the development of central synapses in the mammalian brain.

A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis.

The exocytosis of synaptic vesicles (SVs) elicited by potent stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles ('bulk' endosomes). Subsequently, these vacuoles disappear in parallel with the reappearance of new SVs. We have used synapses of dynamin 1 and 3 double knock-out neurons, where clathrin-mediated endocytosis (CME) is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not defective, but rather enhanced, in the absence of dynamin 1 and 3. The subsequent conversion of bulk endosomes into SVs was not accompanied by the accumulation of clathrin coated buds on their surface and this process proceeded even after further clathrin knock-down, suggesting its independence of clathrin. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin 1/3 and that operates during intense synaptic activity.

Dynamin 1 depletion and memory deficits in rats treated with Abeta and cerebral ischemia.

Alzheimer's disease (AD) is progressive dementia with senile plaques composed of beta-amyloid (Abeta). Recent studies suggest that synaptic dysfunction is one of the earliest events in the pathogenesis of AD. Here we provide the first experimental evidence that a change in the level of dynamin 1 induced by Abeta correlates with memory impairment in vivo. We treated rats with transient cerebral ischemia with oligomeric forms of Abeta (Abeta oligomers), including dimers, trimers, and tetramers, intracerebroventricularly. The combination of Abeta oligomers and cerebral ischemia, but not cerebral ischemia alone, significantly impaired memory and decreased the level of dynamin 1, which plays a critical role in synaptic vesicle recycling, but did not affect the levels of other synaptic proteins, such as synaptophysin and synaptobrevin, in the hippocampus. Furthermore, the N-methyl-D-aspartate (NMDA) receptor antagonist memantine prevented memory impairment and dynamin 1 degradation, suggesting that these changes might be mediated by NMDA receptors. These results suggest that Abeta oligomers induce memory impairment via dynamin 1 degradation, which may imply that dynamin 1 degradation is one of the causes of synaptic dysfunction in AD.