Clusterin expression is upregulated following acute head injury and localizes to astrocytes in old head injury.

There is mounting evidence linking traumatic brain injury (TBI) to neurodegeneration. Clusterin (apolipoprotein J or ApoJ) is a complement inhibitor that appears to have a neuroprotective effect in response to tissue damage and has been reported to be upregulated in Alzheimer's disease. Here we investigated the time course and cellular expression pattern of clusterin in human TBI. Tissue from 32 patients with TBI of varying survival times (from under 30 min to 10 months) was examined using immunohistochemistry for clusterin alongside other markers of neurodegeneration and neuroinflammation. TBI cases were compared to ischemic brain damage, Alzheimer's disease and controls. Double immunofluorescence was carried out in order to examine cellular expression. Clusterin was initially expressed in an axonal location less than 30 min following TBI and increased in intensity and the frequency of deposits with increasing survival time up to 24 h, after which it appeared to reduce in intensity but was still evident several weeks after injury. Clusterin was first evident in astrocytes after 45 min, being increasingly seen up to 48 h but remaining intense in TBI cases with long survival times. Our results suggest clusterin plays a role in modulating the inflammatory response of acute and chronic TBI and that it is a useful marker for TBI, particularly in cases with short survival times. Its prominent accumulation in astrocytes, alongside a mounting inflammatory response and activation of microglial cells supports a potential role in the neurodegenerative changes that occur as a result of TBI.

Novel mutations support a role for Profilin 1 in the pathogenesis of ALS.

Mutations in the gene encoding profilin 1 (PFN1) have recently been shown to cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. We sequenced the PFN1 gene in a cohort of ALS patients (n = 485) and detected 2 novel variants (A20T and Q139L), as well as 4 cases with the previously identified E117G rare variant (∼ 1.2%). A case-control meta-analysis of all published E117G ALS+/- frontotemporal dementia cases including those identified in this report was significant p = 0.001, odds ratio = 3.26 (95% confidence interval, 1.6-6.7), demonstrating this variant to be a susceptibility allele. Postmortem tissue from available patients displayed classic TAR DNA-binding protein 43 pathology. In both transient transfections and in fibroblasts from a patient with the A20T change, we showed that this novel PFN1 mutation causes protein aggregation and the formation of insoluble high molecular weight species which is a hallmark of ALS pathology. Our findings show that PFN1 is a rare cause of ALS and adds further weight to the underlying genetic heterogeneity of this disease.

Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice.

Senile plaques comprised of Aß aggregates and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau filaments are the hallmarks of Alzheimer's disease (AD). A number of amyloid precursor protein (APP) transgenic (Tg) mice harboring APP mutations have been generated as animal models of AD. These mice successfully display amyloid plaque formation and subsequent tau hyperphosphorylation, but seldom induce NFT formations. We have demonstrated that the APPOSK-Tg mice, which possess the E693Δ (Osaka) mutation in APP and thereby accumulate Aß oligomers without plaques, exhibit tau hyperphosphorylation at 8 months, but not NFT formation even at 24 months. We assumed that APP-Tg mice, including ours, failed to form NFTs because NFT formation requires human tau. To test this hypothesis, we crossbred APPOSK-Tg mice with tau-Tg mice (tau264), which express low levels of 3-repeat and 4-repeat wild-type human tau without any pathology. The resultant double Tg mice displayed tau hyperphosphorylation at 6 months and NFT formation at 18 months in the absence of tau mutations. Importantly, these NFTs contained both 3-repeat and 4-repeat human tau, similar to those in AD. Furthermore, the double Tg mice exhibited Aß oligomer accumulation, synapse loss, and memory impairment at 6 months and neuronal loss at 18 months, all of which appeared earlier than in the parent APPOSK-Tg mice. These results suggest that Aß and human tau synergistically interact to accelerate each other's pathology, that the presence of human tau is critical for NFT formation, and that Aß oligomers can induce NFTs in the absence of amyloid plaques.

Simulated surgical-type cerebral biopsies from post-mortem brains allows accurate neuropathological diagnoses in the majority of neurodegenerative disease groups.

BACKGROUND: In theory, cerebral biopsies could provide the diagnosis in a significant proportion of patients with neurodegenerative diseases, however, there are considerable ethical barriers. Previous series of cerebral biopsies have shown variable diagnostic accuracy but have understandably suffered because of lack of post-mortem tissue with which to compare the diagnosis. To determine the accuracy of such biopsies in neurodegenerative disease we took small biopsy-sized samples of predominantly fresh post-mortem brain tissue from frontal and temporal lobes in 62 cases. These were processed as for a biopsy and stained for H&E, p62, tau, Aß, α-synuclein, and TDP-43. The sections were assessed blind by 3 neuropathologists and the results compared with the final post-mortem diagnosis. RESULTS: The agreement and sensitivity in most cases was good especially: controls; Alzheimer's disease (AD); multiple system atrophy (MSA); frontotemporal lobar degeneration with TDP-43 positive inclusions and/or motor neurone disease (FTLD-TDP/MND); Huntington's disease (HD); corticobasal degeneration (CBD) / microtubular associated protein tau mutation cases with CBD-like features (CBD/MAPT); and combined AD- Dementia with Lewy Bodies (AD-DLB) where the sensitivity on assessing both brain regions varied between 75-100%. There was poor sensitivity for progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) (both 0%), but moderate sensitivity for pure DLB (60%). The temporal lobe assessment was marginally more accurate than the frontal lobe but these were only slightly worse than both combined. CONCLUSIONS: The study shows that with certain caveats the cerebral biopsy in life should be a viable method of accurately diagnosing many neurodegenerative diseases.

Mixed tau, TDP-43 and p62 pathology in FTLD associated with a C9ORF72 repeat expansion and p.Ala239Thr MAPT (tau) variant.

A massive intronic GGGGCC hexanucleotide repeat expansion in C9ORF72 has recently been identified as the most common cause of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We have previously demonstrated that C9ORF72 mutant cases have a specific pathological profile with abundant p62-positive, TDP-43-negative cytoplasmic and intranuclear inclusions within cerebellar granular cells of the cerebellum and pyramidal cells of the hippocampus in addition to classical TDP-43 pathology. Here, we report mixed tau and TDP-43 pathology in a woman with behavioural variant FTLD who had the C9ORF72 mutation, and the p.Ala239Thr variant in MAPT (microtubule associated protein tau) gene not previously associated with tau pathology. Two of her brothers, who carried the C9ORF72 mutation, but not the MAPT variant, developed classical ALS without symptomatic cognitive changes. The dominant neuropathology in this woman with FTLD was a tauopathy with Pick's disease-like features. TDP-43 labelling was mainly confined to Pick bodies, but p62-positive, TDP-43-negative inclusions, characteristic of C9ORF72 mutations, were present in the cerebellum and hippocampus. Mixed pathology to this degree is unusual. One might speculate that the presence of the C9ORF72 mutation might influence tau deposition in what was previously thought to be a "benign" variant in MAPT in addition to the aggregation of TDP-43 and other as yet unidentified proteins decorated with ubiquitin and p62.

An MND/ALS phenotype associated with C9orf72 repeat expansion: abundant p62-positive, TDP-43-negative inclusions in cerebral cortex, hippocampus and cerebellum but without associated cognitive decline.

The transactive response DNA binding protein (TDP-43) proteinopathies describe a clinico-pathological spectrum of multi-system neurodegeneration that spans motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) and frontotemporal lobar degeneration (FTLD). We have identified four male patients who presented with the clinical features of a pure MND/ALS phenotype (without dementia) but who had distinctive cortical and cerebellar pathology that was different from other TDP-43 proteinopathies. All patients initially presented with weakness of limbs and respiratory muscles and had a family history of MND/ALS. None had clinically identified cognitive decline or dementia during life and they died between 11 and 32 months after symptom onset. Neuropathological investigation revealed lower motor neuron involvement with TDP-43-positive inclusions typical of MND/ALS. In contrast, the cerebral pathology was atypical, with abundant star-shaped p62-immunoreactive neuronal cytoplasmic inclusions in the cerebral cortex, basal ganglia and hippocampus, while TDP-43-positive inclusions were sparse. This pattern was also seen in the cerebellum where p62-positive, TDP-43-negative inclusions were frequent in granular cells. Western blots of cortical lysates, in contrast to those of sporadic MND/ALS and FTLD-TDP, showed high p62 levels and low TDP-43 levels with no high molecular weight smearing. MND/ALS-associated SOD1, FUS and TARDBP gene mutations were excluded; however, further investigations revealed that all four of the cases did show a repeat expansion of C9orf72, the recently reported cause of chromosome 9-linked MND/ALS and FTLD. We conclude that these chromosome 9-linked MND/ALS cases represent a pathological sub-group with abundant p62 pathology in the cerebral cortex, hippocampus and cerebellum but with no significant associated cognitive decline.

p62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS.

Neuronal cytoplasmic inclusions (NCIs) containing phosphorylated TDP-43 (p-TDP-43) are the pathological hallmarks of motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) and FTLD-TDP. The vast majority of NCIs in the brain and spinal cord also label for ubiquitin and p62, however, we have previously reported a subset of TDP-43 proteinopathy patients who have unusual and abundant p62 positive, TDP-43 negative inclusions in the cerebellum and hippocampus. Here we sought to determine whether these cases carry the hexanucleotide repeat expansion in C9orf72. Repeat primer PCR was performed in 36 MND/ALS, FTLD-MND/ALS and FTLD-TDP cases and four controls. Fourteen individuals with the repeat expansion were detected. In all the 14 expansion mutation cases there were abundant globular and star-shaped p62 positive NCIs in the pyramidal cell layer of the hippocampus, the vast majority of which were p-TDP-43 negative. p62 positive NCIs were also abundant in the cerebellar granular and molecular layers in all cases and in Purkinje cells in 12/14 cases but they were only positive for p-TDP-43 in the granular layer of one case. Abundant p62 positive, p-TDP-43 negative neuronal intranuclear inclusions (NIIs) were seen in 12/14 cases in the pyramidal cell layer of the hippocampus and in 6/14 cases in the cerebellar granular layer. This unusual combination of inclusions appears pathognomonic for C9orf72 repeat expansion positive MND/ALS and FTLD-TDP which we believe form a pathologically distinct subset of TDP-43 proteinopathies. Our results suggest that proteins other than TDP-43 are binding p62 and aggregating in response to the mutation which may play a mechanistic role in neurodegeneration.

Ubiquitinated, p62 immunopositive cerebellar cortical neuronal inclusions are evident across the spectrum of TDP-43 proteinopathies but are only rarely additionally immunopositive for phosphorylation-dependent TDP-43.

Frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP), frontotemporal lobar degeneration with motor neuron disease/amyotrophic lateral sclerosis (FTLD-MND/ALS) and MND/ALS are thought to represent a clinicopathological spectrum of TDP-43 proteinopathies. The cerebellum has been little studied in these conditions, probably because of the lack of cerebellar signs in most cases. We examined p62 immunohistochemistry on cerebellar sections from 43 TDP-43 proteinopathies (including cases of FTLD-TDP, FTLD-MND/ALS and MND/ALS) together with 72 cases of other neurodegenerative diseases, seven controls and three other disease conditions. In 11 of the TDP-43 proteinopathies (26%) there were numerous p62-positive cerebellar inclusions, predominantly within the granular layer, but also the molecular and Purkinje cell layer. Furthermore, only one of the remaining 82 cases (a familial tauopathy) showed similar p62 positivity. Immunohistochemistry for ubiquitin was positive in the granular layer inclusions. The immunohistochemistry for phosphorylation-independent TDP-43, hyperphosphorylated tau, α-synuclein, fusion sarcoma protein (FUS), and neurofilament was negative. In only one case (a case of FTLD-TDP) were the inclusions positive for phosphorylation-dependent TDP43 (p-TDP-43). Those TDP-43 proteinopathy cases that showed the cerebellar inclusions also tended to display other common features, such as a notable excess of p62 pathology when compared to TDP-43 pathology, especially within the pyramidal neurones of the hippocampus but also in some cases within the neocortex. The results suggest that p62-positive inclusions within the cerebellum are seen in a proportion of cases across the range of the TDP-43 proteinopathy spectrum and they appear to be relatively specific for this group of diseases. The question as to whether these cerebellar-positive cases represent a distinct subgroup remains to be answered. Furthermore, the relationship of the p62 positivity in the cerebellum to the underlying pathological processes awaits to be established.

Abnormal TDP-43 expression is identified in the neocortex in cases of dementia pugilistica, but is mainly confined to the limbic system when identified in high and moderate stages of Alzheimer's disease.

The transactive response (TAR) DNA binding protein TDP-43 has been discovered to be a major ubiquitinated protein in frontotemporal lobar degeneration with ubiquitinated tau-negative inclusions (FTLD-U), which consequently has been renamed FTLD-TDP. However, TDP-43 has since been detected in conditions such as Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) but is often confined to the limbic region rather than the more widespread pattern seen in FTLD-TDP. Previous work has suggested some relationship between hippocampal sclerosis and TDP-43 expression. A number of AD cases of both moderate and high stage were examined to determine whether the pattern of TDP-43 immunohistochemical expression differed and whether any relationship to hippocampal sclerosis could be detected. Cases of hippocampal sclerosis from surgical epilepsy specimens were examined to determine whether hippocampal sclerosis alone could cause abnormal TDP-43 expression. To establish whether abnormal TDP-43 expression in other neurodegenerative diseases resembled the pattern and distribution in FTLD-TDP we examined multiple blocks from a variety of neurodegenerative conditions. In 75% of cases of high-stage AD there was abnormal TDP-43 positivity compared to 57% of moderate-stage AD. While the abnormal TDP-43 positivity was confined to the limbic regions in the moderate stages, occasional cases in the high stages showed neocortical positivity. Also amygdala and/or entorhinal positivity appeared to precede positivity in the dentate gyrus. No relationship could be established between abnormal TDP-43 expression and degree of hippocampal sclerosis either in the surgical or autopsy cases. The pattern of distribution of TDP-43 inclusions from cases of dementia pugilistica most closely resembled that in FTLD-TDP. This raises the question as to whether there may be some shared pathogenic mechanisms between the two conditions.

TDP-43 is consistently co-localized with ubiquitinated inclusions in sporadic and Guam amyotrophic lateral sclerosis but not in familial amyotrophic lateral sclerosis with and without SOD1 mutations.

The transactive response (TAR) DNA binding protein 43 (TDP-43) has been recently implicated as a major component of ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS, motor neuron disease: MND) and ALS-related disorders. In this study, we examined abnormal TDP-43 pathology in 13 sporadic ALS (SALS), six familial ALS (FALS) with and without Cu/Zn superoxide dismutase (SOD1) mutations (SOD1-FALS and non-SOD1-FALS), Guam ALS, two frontotemporal lobar degeneration with MND/ALS (FTLD-MND/ALS), one FTLD with ubiquitin-only-immunoreactive inclusions (FTLD-U) and two progressive supranuclear palsy (PSP). Sections from the spinal cord were processed for immunohistochemistry using antibodies against TDP-43, ubiquitin, p62, cystatin C, phosphorylated tau protein (P-tau; AT8), alpha-synuclein and phosphorylated neurofilament protein (P-NF). In 12 out of 13 SALS and both Guam ALS cases ubiquitin and p62-immunoreactive (IR) neuronal inclusions co-localized with TDP-43. In three out of four SOD1-FALS and one of two non-SOD1-FALS cases, TDP-43-IR inclusions were absent despite the presence of p62 and/or ubiquitin-IR inclusions. However, a single TDP-43-IR neuronal inclusion co-localized with p62 and ubiquitin in one SOD1-FALS (His48Gln) case. Except for one neuron in a Guam case, all TDP-43-IR neuronal inclusions were negative for P-tau (AT8). TDP-43-IR glial inclusions and neurites were also demonstrated. The TDP-43 is a consistent component of the ubiquitinated inclusions in SALS and Guam ALS, but TDP-43-IR inclusions are absent or scarce in SOD1-FALS.